Plant regulatory elements and uses thereof

ABSTRACT

The invention provides recombinant DNA molecules and constructs, as well as their nucleotide sequences, useful for modulating gene expression in plants. The invention also provides transgenic plants, plant cells, plant parts, and seeds comprising the recombinant DNA molecules operably linked to heterologous transcribable DNA molecules, as are methods of their use.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/448,019, filed Jan. 19, 2017, which is herein incorporated byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The computer readable form of the sequence listing that is contained inthe file named “MONS436US-sequence_listing.txt” is 59,917 bytes (asmeasured in Microsoft Windows®) and was created on Jan. 12, 2018, isfiled by electronic submission concurrently with this application and isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of plant molecular biology and plantgenetic engineering. More specifically, the invention relates to DNAmolecules useful for modulating gene expression in plants.

BACKGROUND

Regulatory elements are genetic elements that regulate gene activity bymodulating the transcription of an operably linked transcribable DNAmolecule. Such elements may include promoters, leaders, introns, and 3′untranslated regions and are useful in the field of plant molecularbiology and plant genetic engineering.

SUMMARY OF THE INVENTION

The invention provides novel synthetic gene regulatory elements for usein plants. The invention also provides recombinant DNA molecules andconstructs comprising the regulatory elements. The present inventionalso provides transgenic plant cells, plants, and seeds comprising thesynthetic regulatory elements. In one embodiment, the syntheticregulatory elements are operably linked to a heterologous transcribableDNA molecule. The present invention also provides methods of using thesynthetic regulatory elements and methods of making and using therecombinant DNA molecules comprising the synthetic regulatory elementsand transgenic plant cells, plants, and seeds comprising the syntheticregulatory elements operably linked to a transcribable DNA molecule.

Thus, in one aspect, the invention provides a recombinant DNA moleculecomprising a DNA sequence selected from the group consisting of: (a) asequence with at least 85 percent sequence identity to any of SEQ IDNOs:1-29 and 43-45; (b) a sequence comprising any of SEQ ID NOs:1-29 and43-45; and (c) a fragment of any of SEQ ID NOs:1-29 and 43-45, whereinthe fragment has gene-regulatory activity; wherein the sequence isoperably linked to a heterologous transcribable DNA molecule. By“heterologous transcribable DNA molecule,” it is meant that thetranscribable DNA molecule is heterologous with respect to thepolynucleotide sequence to which it is operably linked. In specificembodiments, the recombinant DNA molecule comprises a DNA sequencehaving at least about 90 percent, at least 91 percent, at least 92percent, at least 93 percent, at least 94 percent, at least 95 percent,at least 96 percent, at least 97 percent, at least 98 percent, or atleast 99 percent sequence identity to the DNA sequence of any of SEQ IDNOs:1-29 and 43-45. In particular embodiments, the DNA sequencecomprises a regulatory element. In some embodiments the regulatoryelement comprises a promoter. In still other embodiments, theheterologous transcribable DNA molecule comprises a gene of agronomicinterest, such as a gene capable of providing herbicide resistance inplants, or a gene capable of providing plant pest resistance in plants.In still other embodiments, the invention provides a constructcomprising a recombinant DNA molecule as provided herein.

In another aspect, provided herein are transgenic plant cells comprisinga recombinant DNA molecule comprising a DNA sequence selected from thegroup consisting of: (a) a sequence with at least about 85 percentsequence identity to any of SEQ ID NOs: 1-29 and 43-45; (b) a sequencecomprising any of SEQ ID NOs:1-29 and 43-45; and (c) a fragment of anyof SEQ ID NOs:1-29 and 43-45, wherein the fragment has gene-regulatoryactivity; wherein the DNA sequence is operably linked to a heterologoustranscribable DNA molecule. In certain embodiments, the transgenic plantcell is a monocotyledonous plant cell. In other embodiments, thetransgenic plant cell is a dicotyledonous plant cell.

In still yet another aspect, further provided herein is a transgenicplant, or part thereof, comprising a recombinant DNA molecule comprisinga DNA sequence selected from the group consisting of: a) a sequence withat least 85 percent sequence identity to any of SEQ ID NOs:1-29 and43-45; b) a sequence comprising any of SEQ ID NOs:1-29 and 43-45; and c)a fragment of any of SEQ ID NOs:1-29 and 43-45, wherein the fragment hasgene-regulatory activity; wherein the sequence is operably linked to aheterologous transcribable DNA molecule. In specific embodiments, thetransgenic plant is a progeny plant of any generation that comprises therecombinant DNA molecule. A transgenic seed comprising the recombinantDNA molecule that produces such a transgenic plant when grown is alsoprovided herein.

In another aspect, the invention provides a method of producing acommodity product comprising obtaining a transgenic plant or partthereof containing a recombinant DNA molecule of the invention andproducing the commodity product therefrom. In one embodiment, thecommodity product is processed seeds, grains, plant parts, oils andmeal.

In still yet another aspect, the invention provides a method ofproducing a transgenic plant comprising a recombinant DNA molecule ofthe invention comprising transforming a plant cell with the recombinantDNA molecule of the invention to produce a transformed plant cell andregenerating a transgenic plant from the transformed plant cell.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a DNA sequence of a synthetic regulatory expressionelements group (EXP), EXP-At.GSP442.nno+At.Cyco:3 comprising a syntheticpromoter (P-At.GSP442.nno:2), operably linked 5′ to a synthetic leader(L-At.GSP442.nno:1), operably linked 5′ to an intron (I-At.Cyco:2).

SEQ ID NO:2 is a synthetic promoter sequence, P-At.GSP442.nno:2.

SEQ ID NO:3 is a synthetic leader sequence, L-At.GSP442.nno:1.

SEQ ID NO:4 is a DNA sequence of a synthetic EXP, EXP-At.GSP571comprising a synthetic promoter (P-At.GSP571.nno:5), operably linked 5′to a synthetic leader (L-At.GSP571.nno:1).

SEQ ID NO:5 is a synthetic promoter sequence, P-At.GSP571.nno:5.

SEQ ID NO:6 is a synthetic leader sequence, L-At.GSP571.nno:1.

SEQ ID NO:7 is a DNA sequence of a synthetic regulatory expressionelements group (EXP), EXP-At.GSP571.nno+At.Cyco:2 comprising a syntheticpromoter (P-At.GSP571.nno:5), operably linked 5′ to a synthetic leader(L-At.GSP571.nno:1), operably linked 5′ to an intron (I-At.Cyco:2).

SEQ ID NO:8 is a DNA sequence of a synthetic regulatory expressionelements group (EXP), EXP-At.GSP571.nno+At.GSI21.nno:10 comprising asynthetic promoter (P-At.GSP571.nno:5), operably linked 5′ to asynthetic leader (L-At.GSP571.nno:1), operably linked 5′ to a syntheticintron (I-At.GSI21.nno:2).

SEQ ID NO:9 is a synthetic intron sequence, I-At.GSI21.nno:2.

SEQ ID NO:10 is a DNA sequence of a synthetic EXP,EXP-At.GSP571.nno+At.GSI102.nno:1 comprising a synthetic promoter(P-At.GSP571.nno:5), operably linked 5′ to a synthetic leader(L-At.GSP571.nno:1), operably linked 5′ to a synthetic intron(I-At.GSI102.nno:1).

SEQ ID NO:11 is a synthetic intron sequence, I-At.GSI102.nno:1.

SEQ ID NO: 12 is a DNA sequence of a synthetic EXP, EXP-At.GSP564comprising a synthetic promoter (P-At.GSP564.nno:3), operably linked 5′to a synthetic leader (L-At.GSP564.nno:1).

SEQ ID NO:13 is a synthetic promoter sequence, P-At.GSP564.nno:3.

SEQ ID NO:14 is a synthetic leader sequence, L-At.GSP564.nno:1.

SEQ ID NO:15 is a DNA sequence of a synthetic EXP,EXP-At.GSP564.nno+At.Cyco:2 comprising a synthetic promoter(P-At.GSP564.nno:3), operably linked 5′ to a synthetic leader(L-At.GSP564.nno:1), operably linked 5′ to an intron (I-At.Cyco:2).

SEQ ID NO:16 is a DNA sequence of a synthetic EXP,EXP-At.GSP564.nno+At.GSI17.nno:2 comprising a synthetic promoter(P-At.GSP564.nno:3), operably linked 5′ to a synthetic leader(L-At.GSP564.nno:1), operably linked 5′ to a synthetic intron(I-At.GSI17.nno:1).

SEQ ID NO:17 is a synthetic intron sequence, I-At.GSI17.nno:1.

SEQ ID NO:18 is a DNA sequence of a synthetic EXP,EXP-At.GSP564.nno+At.GSI102.nno:1 comprising a synthetic promoter(P-At.GSP564.nno:3), operably linked 5′ to a synthetic leader(L-At.GSP564.nno:1), operably linked 5′ to a synthetic intron(I-At.GSI102.nno:1).

SEQ ID NO:19 is a DNA sequence of a synthetic EXP, EXP-At.GSP579comprising a synthetic promoter (P-At.GSP579.nno:2), operably linked 5′to a synthetic leader (L-At.GSP579.nno:1).

SEQ ID NO:20 is a synthetic promoter sequence, P-At.GSP579.nno:2.

SEQ ID NO:21 is a synthetic leader sequence, L-At.GSP579.nno:1.

SEQ ID NO:22 is a DNA sequence of a synthetic EXP,EXP-At.GSP579.nno+At.GSI102.nno:3 comprising a synthetic promoter(P-At.GSP579.nno:2), operably linked 5′ to a synthetic leader(L-At.GSP579.nno:1), operably linked 5′ to synthetic intron(I-At.GSI102.nno:1).

SEQ ID NO:23 is a DNA sequence of a synthetic EXP,EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 comprising a syntheticchimeric promoter (P-At.GSP571/442, which is comprised of a syntheticenhancer (E-At.GSP571.nno:1) operably linked 5′ to a synthetic promoter(P-At.GSP442.nno:2)), operably linked 5′ to a synthetic leader(L-At.GSP442.nno:1), operably linked 5′ to a leader (L-At.Cyco-1:1:2),operably linked 5′ to an intron (I-At.Cyco:2).

SEQ ID NO:24 is a synthetic enhancer sequence, E-At.GSP571.nno:1.

SEQ ID NO:25 is a DNA sequence of a synthetic chimeric promoter,P-At.GSP571/442 comprised of a synthetic enhancer (E-At.GSP571.nno:1)operably linked 5′ to a synthetic promoter (P-At.GSP442.nno:2).

SEQ ID NO:26 is a DNA sequence of a synthetic EXP,EXP-At.GSP576.nno+At.GSI17.nno:3 comprising a synthetic promoter(P-At.GSP576.nno:4), operably linked 5′ to a synthetic leader(L-At.GSP576.nno:2), operably linked 5′ to synthetic intron(I-At.GSI17.nno:1).

SEQ ID NO:27 is a synthetic promoter sequence, P-At.GSP576.nno:4.

SEQ ID NO:28 is a synthetic leader sequence, L-At.GSP576.nno:2.

SEQ ID NO:29 is a synthetic 3′ UTR, T-Zm.GST59.nno:1.

SEQ ID NO:30 is a DNA sequence of a synthetic EXP,EXP-At.GSP221+At.Cyco:3 comprising a synthetic promoter (P-At.GSP221:3),operably linked 5′ to a synthetic leader (L-At.GSP221:1), operablylinked 5′ to an intron (I-At.Cyco:2).

SEQ ID NO:31 is a synthetic promoter sequence, P-At.GSP221:3.

SEQ ID NO:32 is a synthetic leader sequence, L-At.GSP221:1.

SEQ ID NO:33 is an intron sequence, I-At.Cyco:2 derived from aCytochrome c oxidase subunit VIa gene from Arabidopsis.

SEQ ID NO:34 is a 3′ UTR sequence, T-Mt.Sali3-2-1:2:1 derived from theSali3 gene of Medicago truncatula.

SEQ ID NO:35 is a 3′ UTR sequence, T-Mt.Oxr-1:2:1 derived from aputative oxidoreductase (OXR) protein gene from Medicago truncatula.

SEQ ID NO:36 is a 3′ UTR sequence, T-Gb.FbL2:1 derived from theGossypium barbadense FbLate-2 gene.

SEQ ID NO:37 is a 3′ UTR sequence, T-Mt.RD22-1:2:1 derived from adehydration-responsive protein RD22 gene from Medicago truncatula.

SEQ ID NO:38 is a DNA sequence of an EXP derived from a Cytochrome coxidase subunit VIa gene from Arabidopsis, EXP-At.Cyco:1:1 comprising apromoter (P-At.Cyco-1:1:2), operably linked 5′ to a leader(L-At.Cyco-1:1:2), operably linked 5′ to intron (I-At.Cyco-1:1:1).

SEQ ID NO:39 is a promoter sequence, P-At.Cyco-1:1:2 derived from aCytochrome c oxidase subunit VIa gene from Arabidopsis.

SEQ ID NO:40 is a leader sequence, L-At.Cyco-1:1:2 derived from aCytochrome c oxidase subunit VIa gene from Arabidopsis.

SEQ ID NO:41 is an intron sequence, I-At.Cyco-1:1:1 derived from aCytochrome c oxidase subunit VIa gene from Arabidopsis.

SEQ ID NO:42 is a coding sequence for β-glucuronidase (GUS) with aprocessable intron derived from the potato light-inducibletissue-specific ST-LS1 gene (Genbank Accession: X04753).

SEQ ID NO:43 is a DNA sequence of an EXP, EXP-At.GSP442+L-I-At.Cycocomprising the synthetic promoter, P-At.GSP442.nno:2, operably linked 5′to the synthetic leader, L-At.GSP442.nno:1, operably linked 5′ to theleader, L-At.Cyco-1:1:2, which is operably linked 5′ to the intron,I-At.Cyco:2.

SEQ ID NO:44 is a DNA sequence of the synthetic 3′ UTR, T-Zm.GST7.nno:2.

SEQ ID NO:45 is a DNA sequence of an EXP, EXP-At.GSP576.nno+At.Cyco:1comprising the synthetic promoter, P-At.GSP564.nno:3, operably linked 5′to the synthetic leader, L-At.GSP564.nno:1, which is operably linked 5′to the intron, I-At.Cyco:2.

SEQ ID NO:46 is a DNA sequence of the EXP, EXP-CaMV.35S comprising the35S promoter and leader derived from the Cauliflower mosaic virus.

SEQ ID NO:47 is a DNA sequence of the intron, I-Zm.DnaK:1, derived fromthe heat shock protein 70 (Hsp70) gene (DnaK) from Zea mays.

SEQ ID NO:48 is a DNA sequence of the 3′ UTR, T-Os.LTP:1, derived fromthe Lipid Transfer Protein-like gene (LTP) from Oryza sativa.

SEQ ID NO:49 is a coding sequence for the NanoLuc® luciferasefluorescent protein (Promega, Madison, Wis. 53711), Nluc which wasengineered by directed evolution from a deep-sea shrimp (Oplophorusgacilirostris) luciferase.

SEQ ID NO:50 is a DNA sequence of the EXP, EXP-At.Bglu21+At.Cyco:2comprising the promoter and leader of a beta-glucuronidase 21 gene fromArabidopsis thaliana, operably linked 5′ to the intron, I-At.Cyco-1:1:1.

SEQ ID NO:51 is a DNA sequence of the EXP, EXP-CaMV.35S-enh+Ph.DnaK:1:3comprising an enhanced Cauliflower mosaic virus 35S promoter, operablylinked 5′ to the leader of the heat shock protein 70 (HSP70) gene fromPetunia x hybrid.

SEQ ID NO:52 is a DNA sequence of the EXP, EXP-Gm.Sphas1:1:1 comprisingthe promoter and leader of the 7S alpha prime gene of soybean.

SEQ ID NO:53 is a DNA sequence of the EXP, EXP-CaMV.35S-enh+Zm.DnaK:1:1comprising an enhanced Cauliflower mosaic virus 35S promoter, operablylinked 5′ to the intron, I-Zm.DnaK:1.

SEQ ID NO:54 is a DNA sequence encoding a luciferase protein(LUCIFERASE:1:3) derived from Photinus pyralis (Firefly).

SEQ ID NO:55 is a DNA sequence of the 3′ UTR, T-AGRtu.nos-1:1:13 derivedfrom the Agrobacterium tumefaciens nopaline synthase gene.

SEQ ID NO:56 is a DNA sequence of the EXP, EXP-CaMV.35S-enh-Lhcb1comprising an enhanced Cauliflower mosaic virus 35S promoter, operablylinked 5′ to the leader of a chlorophyll a/b-binding gene of thelight-harvesting complex of Triticum aestivum (Wheat).

SEQ ID NO:57 is a DNA sequence encoding a luciferase protein(CR-Ren.hRenilla Lucife-0:0:1) derived from Renilla reniformis (SeaPansy).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides synthetic regulatory elements havinggene-regulatory activity in plants. The nucleotide sequences of thesesynthetic regulatory elements are provided as SEQ ID NOs:1-29 and SEQ IDNOs:43-45. These synthetic regulatory elements are capable of affectingthe expression of an operably linked transcribable DNA molecule in planttissues, and therefore regulating gene expression of an operably linkedtransgene in transgenic plants. The invention also provides methods ofmodifying, producing, and using recombinant DNA molecules which containthe provided synthetic regulatory elements. The invention also providescompositions that include transgenic plant cells, plants, plant parts,and seeds containing the recombinant DNA molecules of the invention, andmethods for preparing and using the same.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

DNA Molecules

As used herein, the term “DNA” or “DNA molecule” refers to adouble-stranded DNA molecule of genomic or synthetic origin, i.e., apolymer of deoxyribonucleotide bases or a DNA molecule, read from the 5′(upstream) end to the 3′ (downstream) end. As used herein, the term “DNAsequence” refers to the nucleotide sequence of a DNA molecule. Thenomenclature used herein corresponds to that of Title 37 of the UnitedStates Code of Federal Regulations § 1.822, and set forth in the tablesin WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

As used herein, a “recombinant DNA molecule” is a DNA moleculecomprising a combination of DNA molecules that would not naturally occurtogether without human intervention. For instance, a recombinant DNAmolecule may be a DNA molecule that is comprised of at least two DNAmolecules heterologous with respect to each other, a DNA molecule thatcomprises a DNA sequence that deviates from DNA sequences that exist innature, a DNA molecule that comprises a synthetic DNA sequence or a DNAmolecule that has been incorporated into a host cell's DNA by genetictransformation or gene editing.

As used herein, a “synthetic nucleotide sequence” or “artificialnucleotide sequence” is a nucleotide sequence that is not known to occurin nature, that is not naturally occurring, or that does not occurwithout human intervention. The gene-regulatory elements of the presentinvention comprise synthetic nucleotide sequences. Preferably, syntheticnucleotide sequences share little or no extended homology to naturalsequences. Extended homology in this context generally refers to 100%sequence identity extending beyond about 25 nucleotides of contiguoussequence.

Reference in this application to an “isolated DNA molecule,” or anequivalent term or phrase, is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. In one embodiment, the term “isolated” refers to a DNA moleculethat is at least partially separated from some of the nucleic acidswhich normally flank the DNA molecule in its native or natural state.Thus, DNA molecules fused to regulatory or coding sequences with whichthey are not normally associated, for example as the result ofrecombinant techniques, are considered isolated herein. Such moleculesare considered isolated when integrated into the chromosome of a hostcell or present in a nucleic acid solution with other DNA molecules, inthat they are not in their native state. For the purposes of thisdisclosure, any transgenic nucleotide sequence, i.e., the nucleotidesequence of the DNA inserted into the genome of the cells of a plant orbacterium, or present in an extrachromosomal vector, would be consideredto be an isolated nucleotide sequence whether it is present within theplasmid or similar structure used to transform the cells, within thegenome of the plant or bacterium, or present in detectable amounts intissues, progeny, biological samples or commodity products derived fromthe plant or bacterium.

As used herein, the term “sequence identity” refers to the extent towhich two optimally aligned polynucleotide sequences or two optimallyaligned polypeptide sequences are identical. An optimal sequencealignment is created by manually aligning two sequences, e.g., areference sequence and another sequence, to maximize the number ofnucleotide matches in the sequence alignment with appropriate internalnucleotide insertions, deletions, or gaps. As used herein, the term“reference sequence” refers to a DNA sequence provided as SEQ IDNOs:1-29 and SEQ ID NOs:43-45.

As used herein, the term “percent sequence identity” or “percentidentity” or “% identity” is the identity fraction multiplied by 100.The “identity fraction” for a sequence optimally aligned with areference sequence is the number of nucleotide matches in the optimalalignment, divided by the total number of nucleotides in the referencesequence, e.g., the total number of nucleotides in the full length ofthe entire reference sequence. Thus, one embodiment of the inventionprovides a DNA molecule comprising a sequence that, when optimallyaligned to a reference sequence, provided herein as any of SEQ IDNOs:1-29 and SEQ ID NOs:43-45, has at least about 85 percent identity,at least about 86 percent identity, at least about 87 percent identity,at least about 88 percent identity, at least about 89 percent identity,at least about 90 percent identity, at least about 91 percent identity,at least about 92 percent identity, at least about 93 percent identity,at least about 94 percent identity, at least about 95 percent identity,at least about 96 percent identity, at least about 97 percent identity,at least about 98 percent identity, at least about 99 percent identity,or at least about 100 percent identity to the reference sequence. Instill further specific embodiments, a sequence having a percent identityto any of SEQ ID NOs:1-29 and SEQ ID NOs:43-45 may be defined asexhibiting promoter activity possessed by the starting sequence fromwhich it is derived. A sequence having a percent identity to any of SEQID NOs:1-29 and SEQ ID NOs:43-45 may further comprise a “minimalpromoter” which provides a basal level of transcription and is comprisedof a TATA box or equivalent sequence for recognition and binding of theRNA polymerase II complex for initiation of transcription.

Regulatory Elements

Regulatory elements such as promoters, leaders (also known as 5′ UTRs),enhancers, introns, and transcription termination regions (or 3′ UTRs)play an integral part in the overall expression of genes in livingcells. The term “regulatory element,” as used herein, refers to a DNAmolecule having gene-regulatory activity. The term “gene-regulatoryactivity,” as used herein, refers to the ability to affect theexpression of an operably linked transcribable DNA molecule, forinstance by affecting the transcription and/or translation of theoperably linked transcribable DNA molecule. Regulatory elements, such aspromoters, leaders, enhancers, introns and 3′ UTRs that function inplants are useful for modifying plant phenotypes through geneticengineering.

As used herein, a “regulatory expression element group” or “EXP”sequence may refer to a group of operably linked regulatory elements,such as enhancers, promoters, leaders, and introns. For example, aregulatory expression element group may be comprised, for instance, of apromoter operably linked 5′ to a leader sequence. EXP's useful inpracticing the present invention include SEQ ID NOs:1, 4, 7, 8, 10, 12,15, 16, 18, 19, 22, 23, 26, 30, 43 and 45.

Regulatory elements may be characterized by their gene expressionpattern, e.g., positive and/or negative effects such as constitutiveexpression or temporal, spatial, developmental, tissue, environmental,physiological, pathological, cell cycle, and/or chemically responsiveexpression, and any combination thereof, as well as by quantitative orqualitative indications. As used herein, a “gene expression pattern” isany pattern of transcription of an operably linked DNA molecule into atranscribed RNA molecule. The transcribed RNA molecule may be translatedto produce a protein molecule or may provide an antisense or otherregulatory RNA molecule, such as a double-stranded RNA (dsRNA), atransfer RNA (tRNA), a ribosomal RNA (rRNA), a microRNA (miRNA), and thelike.

As used herein, the term “protein expression” is any pattern oftranslation of a transcribed RNA molecule into a protein molecule.Protein expression may be characterized by its temporal, spatial,developmental, or morphological qualities, as well as by quantitative orqualitative indications.

A promoter is useful as a regulatory element for modulating theexpression of an operably linked transcribable DNA molecule. As usedherein, the term “promoter” refers generally to a DNA molecule that isinvolved in recognition and binding of RNA polymerase II and otherproteins, such as trans-acting transcription factors, to initiatetranscription. A promoter may be initially isolated from the 5′untranslated region (5′ UTR) of a genomic copy of a gene. Alternately,promoters may be synthetically produced or manipulated DNA molecules.Promoters may also be chimeric. Chimeric promoters are produced throughthe fusion of two or more heterologous DNA molecules. Promoters usefulin practicing the present invention include promoter elements comprisedwithin any of SEQ ID NOs:2, 5, 13, 20, 25, 27, 31 and 39 or fragments orvariants thereof. In specific embodiments of the invention, the claimedDNA molecules and any variants or derivatives thereof as describedherein, are further defined as comprising promoter activity, i.e., arecapable of acting as a promoter in a host cell, such as in a transgenicplant. In still further specific embodiments, a fragment may be definedas exhibiting promoter activity possessed by the starting promotermolecule from which it is derived, or a fragment may comprise a “minimalpromoter” which provides a basal level of transcription and is comprisedof a TATA box or equivalent DNA sequence for recognition and binding ofthe RNA polymerase II complex for initiation of transcription.

In one embodiment, fragments of a promoter sequence disclosed herein areprovided. Promoter fragments may comprise promoter activity, asdescribed above, and may be useful alone or in combination with otherpromoters and promoter fragments, such as in constructing chimericpromoters, or in combination with other expression elements andexpression element fragments. In specific embodiments, fragments of apromoter are provided comprising at least about 50, at least about 75,at least about 95, at least about 100, at least about 125, at leastabout 150, at least about 175, at least about 200, at least about 225,at least about 250, at least about 275, at least about 300, at leastabout 500, at least about 600, at least about 700, at least about 750,at least about 800, at least about 900, or at least about 1000contiguous nucleotides, or longer, of a DNA molecule having promoteractivity as disclosed herein. In certain embodiments, the inventionprovides fragments of a promoter provided herein, having the activity ofthe full length sequence. Methods for producing such fragments from astarting promoter molecule are well known in the art.

Compositions derived from any of the promoter elements comprised withinany of SEQ ID NOs:2, 5, 13, 20, 25, 27, 31 and 39, such as internal or5′ deletions, for example, can be produced using methods known in theart to improve or alter expression, including by removing elements thathave either positive or negative effects on expression; duplicatingelements that have positive or negative effects on expression; and/orduplicating or removing elements that have tissue- or cell-specificeffects on expression. Compositions derived from any of the promoterelements comprised within any of SEQ ID NOs:2, 5, 13, 20, 25, 27, 31 and39 comprised of 3′ deletions in which the TATA box element or equivalentsequence thereof and downstream sequence is removed can be used, forexample, to make enhancer elements. Further deletions can be made toremove any elements that have positive or negative; tissue-specific;cell-specific; or timing-specific (such as, but not limited to,circadian rhythm) effects on expression. Any of the promoter elementscomprised within any of SEQ ID NOs:2, 5, 13, 20, 25, 27, 31 and 39 andfragments or enhancers derived therefrom can be used to make chimerictranscriptional regulatory element compositions.

In accordance with the invention, a promoter or promoter fragment may beanalyzed for the presence of known promoter elements, i.e., DNA sequencecharacteristics, such as a TATA box and other known transcription factorbinding site motifs. Identification of such known promoter elements maybe used by one of skill in the art to design variants of the promoterhaving a similar expression pattern to the original promoter.

As used herein, the term “leader” refers to a DNA molecule isolated fromthe untranslated 5′ region (5′ UTR) a gene and defined generally as anucleotide segment between the transcription start site (TSS) and theprotein coding sequence start site. Alternately, leaders may besynthetically produced or manipulated DNA elements. A leader can be usedas a 5′ regulatory element for modulating expression of an operablylinked transcribable DNA molecule. Leader molecules may be used with aheterologous promoter or with their native promoter. Leaders useful inpracticing the present invention include SEQ ID NOs:3, 6, 14, 21, 28, 32and 40; or any of the leader elements comprised within any of SEQ IDNOs:1, 4, 7, 8, 10, 12, 15, 16, 18, 19, 22, 23, 26, 30, 43 and 45 orfragments or variants thereof. In specific embodiments, such DNAsequences may be defined as being capable of acting as a leader in ahost cell, including, for example, a transgenic plant cell. In oneembodiment, such sequences are decoded as comprising leader activity.

The leader sequences (also referred to as 5′ UTRs) presented as SEQ IDNOs:3, 6, 14, 21, 28, 32 and 40 or any of the leader elements comprisedwithin any of SEQ ID NOs:1, 4, 7, 8, 10, 12, 15, 16, 18, 19, 22, 23, 26,30 and 43 may be comprised of regulatory elements, or may adoptsecondary structures that can have an effect on transcription ortranslation of an operably linked transcribable DNA molecule. The leadersequences presented as SEQ ID NOs:3, 6, 14, 21, 28, 32 and 40 or any ofthe leader elements comprised within any of SEQ ID NOs:1, 4, 7, 8, 10,12, 15, 16, 18, 19, 22, 23, 26, 30, 43 and 45 can be used in accordancewith the invention to make chimeric regulatory elements that affecttranscription or translation of a an operably linked transcribable DNAmolecule.

As used herein, the term “intron” refers to a DNA molecule that may beisolated or identified from a gene and may be defined generally as aregion spliced out during messenger RNA (mRNA) processing prior totranslation. Alternately, an intron may be a synthetically produced ormanipulated DNA element. An intron may contain enhancer elements thateffect the transcription of operably linked genes. An intron may be usedas a regulatory element for modulating expression of an operably linkedtranscribable DNA molecule. A construct may comprise an intron, and theintron may or may not be heterologous with respect to the transcribableDNA molecule. Examples of introns known in the art include the riceactin intron and the corn HSP70 intron.

In plants, the inclusion of some introns in gene constructs leads toincreased mRNA and protein accumulation relative to constructs lackingthe intron. This effect has been termed “intron mediated enhancement”(IME) of gene expression. Introns known to stimulate expression inplants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, andUbi1), in rice genes (e.g., tpi) and in dicotyledonous plant genes likethose from petunia (e.g., rbcS), potato (e.g., st-ls1) and fromArabidopsis thaliana (e.g., ubq3 and pat1). It has been shown thatdeletions or mutations within the splice sites of an intron reduce geneexpression, indicating that splicing might be needed for IME. However,IME in dicotyledonous plants has been shown by point mutations withinthe splice sites of the pat1 gene from A. thaliana. Multiple uses of thesame intron in one plant have been shown to exhibit disadvantages. Inthose cases, it is necessary to have a collection of basic controlelements for the construction of appropriate recombinant DNA elements.Exemplary introns useful in practicing the present invention arepresented as SEQ ID NOs:9, 11, 17, 33 and 41.

As used herein, the terms “3′ transcription termination molecule,” “3′untranslated region” or “3′ UTR” refer to a DNA molecule that is usedduring transcription to the untranslated region of the 3′ portion of anmRNA molecule. The 3′ untranslated region of an mRNA molecule may begenerated by specific cleavage and 3′ polyadenylation, also known as apolyA tail. A 3′ UTR may be operably linked to and located downstream ofa transcribable DNA molecule and may include a polyadenylation signaland other regulatory signals capable of affecting transcription, mRNAprocessing, or gene expression. PolyA tails are thought to function inmRNA stability and in initiation of translation. Examples of 3′transcription termination molecules in the art are the nopaline synthase3′ region; wheat hsp17 3′ region, pea rubisco small subunit 3′ region,cotton E6 3′ region, and the coixin 3′ UTR.

3′ UTRs typically find beneficial use for the recombinant expression ofspecific DNA molecules. A weak 3′ UTR has the potential to generateread-through, which may affect the expression of the DNA moleculelocated in the neighboring expression cassettes. Appropriate control oftranscription termination can prevent read-through into DNA sequences(e.g., other expression cassettes) localized downstream and can furtherallow efficient recycling of RNA polymerase to improve gene expression.Efficient termination of transcription (release of RNA Polymerase IIfrom the DNA) is prerequisite for re-initiation of transcription andthereby directly affects the overall transcript level. Subsequent totranscription termination, the mature mRNA is released from the site ofsynthesis and template transported to the cytoplasm. Eukaryotic mRNAsare accumulated as poly(A) forms in vivo, making it difficult to detecttranscriptional termination sites by conventional methods. However,prediction of functional and efficient 3′ UTRs by bioinformatics methodsis difficult in that there are no conserved DNA sequences that wouldallow easy prediction of an effective 3′ UTR.

From a practical standpoint, it is typically beneficial that a 3′ UTRused in an expression cassette possesses the following characteristics.First, the 3′ UTR should be able to efficiently and effectivelyterminate transcription of the transgene and prevent read-through of thetranscript into any neighboring DNA sequence, which can be comprised ofanother expression cassette as in the case of multiple expressioncassettes residing in one transfer DNA (T-DNA), or the neighboringchromosomal DNA into which the T-DNA has inserted. Second, the 3′ UTRshould not cause a reduction in the transcriptional activity imparted bythe promoter, leader, enhancers, and introns that are used to driveexpression of the DNA molecule. Finally, in plant biotechnology, the 3′UTR is often used for priming of amplification reactions of reversetranscribed RNA extracted from the transformed plant and used to: (1)assess the transcriptional activity or expression of the expressioncassette once integrated into the plant chromosome; (2) assess the copynumber of insertions within the plant DNA; and (3) assess zygosity ofthe resulting seed after breeding. The 3′ UTR is also used inamplification reactions of DNA extracted from the transformed plant tocharacterize the intactness of the inserted cassette. A 3′ UTR useful inpracticing the present invention is presented as SEQ ID NOs:29, 34, 35,36, 37, and 44.

As used herein, the term “enhancer” or “enhancer element” refers to acis-acting regulatory element, a.k.a. cis-element, which confers anaspect of the overall expression pattern, but is usually insufficientalone to drive transcription, of an operably linked transcribable DNAmolecule. Unlike promoters, enhancer elements do not usually include atranscription start site (TSS) or TATA box or equivalent DNA sequence. Apromoter or promoter fragment may naturally comprise one or moreenhancer elements that affect the transcription of an operably linkedDNA sequence. An enhancer element may also be fused to a promoter toproduce a chimeric promoter cis-element, which confers an aspect of theoverall modulation of gene expression. An example of an enhancer elementderived from the synthetic promoter, P-At.GSP571.nno:5 (SEQ ID NO:5) isprovided as SEQ ID NO:24 (E-At.GSP571.nno:1).

Many promoter enhancer elements are believed to bind DNA-bindingproteins and/or affect DNA topology, producing local conformations thatselectively allow or restrict access of RNA polymerase to the DNAtemplate or that facilitate selective opening of the double helix at thesite of transcriptional initiation. An enhancer element may function tobind transcription factors that regulate transcription. Some enhancerelements bind more than one transcription factor, and transcriptionfactors may interact with different affinities with more than oneenhancer domain. Enhancer elements can be identified by a number oftechniques, including deletion analysis, i.e., deleting one or morenucleotides from the 5′ end or internal to a promoter; DNA bindingprotein analysis using DNase I footprinting; methylation interference;electrophoresis mobility-shift assays; in vivo genomic footprinting byligation-mediated polymerase chain reaction (PCR); and otherconventional assays or by DNA sequence similarity analysis using knowncis-element motifs or enhancer elements as a target sequence or targetmotif with conventional DNA sequence comparison methods, such as BLAST.The fine structure of an enhancer domain can be further studied bymutagenesis (or substitution) of one or more nucleotides or by otherconventional methods known in the art. Enhancer elements can be obtainedby chemical synthesis or by isolation from regulatory elements thatinclude such elements, and they can be synthesized with additionalflanking nucleotides that contain useful restriction enzyme sites tofacilitate subsequence manipulation. Thus, the design, construction, anduse of enhancer elements according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the invention. An exemplary enhancer useful inpracticing this invention is presented as SEQ ID NO:24.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither the first nor the second DNA molecule would normally be found inthat configuration, i.e. fused to the other. The chimeric DNA moleculeis thus a new DNA molecule not otherwise normally found in nature. Asused herein, the term “chimeric promoter” refers to a promoter producedthrough such manipulation of DNA molecules. A chimeric promoter maycombine two or more DNA fragments for example, the fusion of a promoterto an enhancer element. Thus, the design, construction, and use ofchimeric promoters according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the present invention. An exemplary chimeric promoteris presented herein as SEQ ID NO:25 (P-At.GSP571/442).

Chimeric regulatory elements can be designed to comprise variousconstituent elements which may be operatively linked by various methodsknown in the art, such as restriction enzyme digestion and ligation,ligation independent cloning, modular assembly of PCR products duringamplification, or direct chemical synthesis of the regulatory element,as well as other methods known in the art. The resulting variouschimeric regulatory elements can be comprised of the same, or variantsof the same, constituent elements but differ in the DNA sequence or DNAsequences that comprise the linking DNA sequence or sequences that allowthe constituent parts to be operatively linked. In the invention, theDNA sequences provided as SEQ ID NOs:1-29 and SEQ ID NOs:43-45 mayprovide regulatory element reference sequences, wherein the constituentelements that comprise the reference sequence may be joined by methodsknown in the art and may comprise substitutions, deletions, and/orinsertions of one or more nucleotides or mutations that naturally occurin bacterial and plant cell transformation.

As used herein, the term “variant” refers to a second DNA molecule, suchas a regulatory element, that is in composition similar, but notidentical to, a first DNA molecule, and wherein the second DNA moleculestill maintains the general functionality, i.e. the same or similarexpression pattern, for instance through more or less equivalenttranscriptional activity, of the first DNA molecule. A variant may be ashorter or truncated version of the first DNA molecule or an alteredversion of the sequence of the first DNA molecule, such as one withdifferent restriction enzyme sites and/or internal deletions,substitutions, or insertions. A “variant” can also encompass aregulatory element having a nucleotide sequence comprising asubstitution, deletion, or insertion of one or more nucleotides of areference sequence, wherein the derivative regulatory element has moreor less or equivalent transcriptional or translational activity than thecorresponding parent regulatory molecule. In the present invention, apolynucleotide sequence provided as SEQ ID NOs:1-29 and SEQ ID NOs:43-45may be used to create variants that are in similar in composition, butnot identical to, the DNA sequence of the original regulatory element,while still maintaining the general functionality, i.e., the same orsimilar expression pattern, of the original regulatory element.Production of such variants of the invention is well within the ordinaryskill of the art in light of the disclosure and is encompassed withinthe scope of the invention.

The efficacy of the modifications, duplications, or deletions describedherein on the desired expression aspects of a particular transgene maybe tested empirically in stable and transient plant assays, such asthose described in the working examples, so as to validate the results,which may vary depending upon the changes made and the goal of thechange in the starting DNA molecule.

Constructs

As used herein, the term “construct” means any recombinant DNA moleculesuch as a plasmid, cosmid, virus, phage, or linear or circular DNA orRNA molecule, derived from any source, capable of genomic integration orautonomous replication, comprising a DNA molecule where at least one DNAmolecule has been linked to another DNA molecule in a functionallyoperative manner, i.e. operably linked. As used herein, the term“vector” means any construct that may be used for the purpose oftransformation, i.e., the introduction of heterologous DNA or RNA into ahost cell. A construct typically includes one or more expressioncassettes. As used herein, an “expression cassette” refers to a DNAmolecule comprising at least a transcribable DNA molecule operablylinked to one or more regulatory elements, typically at least a promoterand a 3′ UTR.

As used herein, the term “operably linked” refers to a first DNAmolecule joined to a second DNA molecule, wherein the first and secondDNA molecules are so arranged that the first DNA molecule affects thefunction of the second DNA molecule. The two DNA molecules may or maynot be part of a single contiguous DNA molecule and may or may not beadjacent. For example, a promoter is operably linked to a transcribableDNA molecule if the promoter modulates transcription of thetranscribable DNA molecule of interest in a cell. A leader, for example,is operably linked to DNA sequence when it is capable of affecting thetranscription or translation of the DNA sequence.

The constructs of the invention may be provided, in one embodiment, asdouble tumor-inducing (Ti) plasmid border constructs that have the rightborder (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of theTi plasmid isolated from Agrobacterium tumefaciens comprising a T-DNAthat, along with transfer molecules provided by the A. tumefacienscells, permit the integration of the T-DNA into the genome of a plantcell (see, e.g., U.S. Pat. No. 6,603,061). The constructs may alsocontain the plasmid backbone DNA segments that provide replicationfunction and antibiotic selection in bacterial cells, e.g., anEscherichia coli origin of replication such as ori322, a broad hostrange origin of replication such as oriV or oriRi, and a coding regionfor a selectable marker such as Spec/Strp that encodes for Tn7aminoglycoside adenyltransferase (aadA) conferring resistance tospectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectablemarker gene. For plant transformation, the host bacterial strain isoften A. tumefaciens ABI, C58, or LBA4404, however other strains knownto those skilled in the art of plant transformation can function in theinvention.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the transcribable DNA molecule istranscribed into a functional mRNA molecule that is translated andexpressed as a protein. For the practice of the invention, conventionalcompositions and methods for preparing and using constructs and hostcells are well known to one skilled in the art. Typical vectors usefulfor expression of nucleic acids in higher plants are well known in theart and include vectors derived from the Ti plasmid of Agrobacteriumtumefaciens and the pCaMVCN transfer control vector.

Various regulatory elements may be included in a construct, includingany of those provided herein. Any such regulatory elements may beprovided in combination with other regulatory elements. Suchcombinations can be designed or modified to produce desirable regulatoryfeatures. In one embodiment, constructs of the invention comprise atleast one regulatory element operably linked to a transcribable DNAmolecule operably linked to a 3′ UTR.

Constructs of the invention may include any promoter or leader providedherein or known in the art. For example, a promoter of the invention maybe operably linked to a heterologous non-translated 5′ leader such asone derived from a heat shock protein gene. Alternatively, a leader ofthe invention may be operably linked to a heterologous promoter such asthe Cauliflower Mosaic Virus 35S transcript promoter.

Expression cassettes may also include a transit peptide coding sequencethat encodes a peptide that is useful for sub-cellular targeting of anoperably linked protein, particularly to a chloroplast, leucoplast, orother plastid organelle; mitochondria; peroxisome; vacuole; or anextracellular location. Many chloroplast-localized proteins areexpressed from nuclear genes as precursors and are targeted to thechloroplast by a chloroplast transit peptide (CTP). Examples of suchisolated chloroplast proteins include, but are not limited to, thoseassociated with the small subunit (SSU) of ribulose-1,5,-bisphosphatecarboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvestingcomplex protein I and protein II, thioredoxin F, and enolpyruvylshikimate phosphate synthase (EPSPS). Chloroplast transit peptides aredescribed, for example, in U.S. Pat. No. 7,193,133. It has beendemonstrated that non-chloroplast proteins may be targeted to thechloroplast by the expression of a heterologous CTP operably linked tothe transgene encoding a non-chloroplast proteins.

Transcribable DNA Molecules

As used herein, the term “transcribable DNA molecule” refers to any DNAmolecule capable of being transcribed into a RNA molecule, including,but not limited to, those having protein coding sequences and thoseproducing RNA molecules having sequences useful for gene suppression.The type of DNA molecule can include, but is not limited to, a DNAmolecule from the same plant, a DNA molecule from another plant, a DNAmolecule from a different organism, or a synthetic DNA molecule, such asa DNA molecule containing an antisense message of a gene, or a DNAmolecule encoding an artificial, synthetic, or otherwise modifiedversion of a transgene. Exemplary transcribable DNA molecules forincorporation into constructs of the invention include, e.g., DNAmolecules or genes from a species other than the species into which theDNA molecule is incorporated or genes that originate from, or arepresent in, the same species, but are incorporated into recipient cellsby genetic engineering methods rather than classical breedingtechniques.

A “transgene” refers to a transcribable DNA molecule heterologous to ahost cell at least with respect to its location in the host cell genomeand/or a transcribable DNA molecule artificially incorporated into ahost cell's genome in the current or any prior generation of the cell.

A regulatory element, such as a synthetic promoter of the invention, maybe operably linked to a heterologous transcribable DNA molecule. As usedherein, the term “heterologous” refers to the combination of two or moreDNA molecules when such a combination is not normally found in nature.For example, the two DNA molecules may be derived from different speciesand/or the two DNA molecules may be derived from different genes, e.g.,different genes from the same species or the same genes from differentspecies, or one of the DNA molecules might be synthetic and not found innature. A regulatory element is heterologous with respect to an operablylinked transcribable DNA molecule if such a combination is not normallyfound in nature, i.e., the transcribable DNA molecule does not naturallyoccur operably linked to the regulatory element.

The transcribable DNA molecule may generally be any DNA molecule forwhich expression of a transcript is desired. Such expression of atranscript may result in translation of the resulting mRNA molecule, andthus protein expression. Alternatively, for example, a transcribable DNAmolecule may be designed to ultimately cause decreased expression of aspecific gene or protein. In one embodiment, this may be accomplished byusing a transcribable DNA molecule that is oriented in the antisensedirection. One of ordinary skill in the art is familiar with using suchantisense technology. Any gene may be negatively regulated in thismanner, and, in one embodiment, a transcribable DNA molecule may bedesigned for suppression of a specific gene through expression of adsRNA, siRNA or miRNA molecule.

Thus, one embodiment of the invention is a recombinant DNA moleculecomprising a regulatory element of the invention, such as those providedas SEQ ID NOs:1-29 and SEQ ID NOs:43-45, operably linked to aheterologous transcribable DNA molecule so as to modulate transcriptionof the transcribable DNA molecule at a desired level or in a desiredpattern when the construct is integrated in the genome of a transgenicplant cell. In one embodiment, the transcribable DNA molecule comprisesa protein-coding region of a gene and in another embodiment thetranscribable DNA molecule comprises an antisense region of a gene.

Genes of Agronomic Interest

A transcribable DNA molecule may be a gene of agronomic interest. Asused herein, the term “gene of agronomic interest” refers to atranscribable DNA molecule that, when expressed in a particular planttissue, cell, or cell type, confers a desirable characteristic. Theproduct of a gene of agronomic interest may act within the plant inorder to cause an effect upon the plant morphology, physiology, growth,development, yield, grain composition, nutritional profile, disease orpest resistance, and/or environmental or chemical tolerance or may actas a pesticidal agent in the diet of a pest that feeds on the plant. Inone embodiment of the invention, a regulatory element of the inventionis incorporated into a construct such that the regulatory element isoperably linked to a transcribable DNA molecule that is a gene ofagronomic interest. In a transgenic plant containing such a construct,the expression of the gene of agronomic interest can confer a beneficialagronomic trait. A beneficial agronomic trait may include, for example,but is not limited to, herbicide tolerance, insect control, modifiedyield, disease resistance, pathogen resistance, modified plant growthand development, modified starch content, modified oil content, modifiedfatty acid content, modified protein content, modified fruit ripening,enhanced animal and human nutrition, biopolymer productions,environmental stress resistance, pharmaceutical peptides, improvedprocessing qualities, improved flavor, hybrid seed production utility,improved fiber production, and desirable biofuel production.

Examples of genes of agronomic interest known in the art include, butare not limited to, those for herbicide resistance (U.S. Pat. Nos.6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775;5,804,425; 5,633,435; and 5,463,175), increased yield (U.S. Pat. Nos.USRE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828;6,399,330; 6,372,211; 6,235,971; 6,222,098; and 5,716,837), insectcontrol (U.S. Pat. Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046;6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655;6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351;6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649;6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756;6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658, 5,880,275;5,763,245; and 5,763,241), fungal disease resistance (U.S. Pat. Nos.6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671;5,773,696; 6,121,436; 6,316,407; and 6,506,962), virus resistance (U.S.Pat. Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and5,304,730), nematode resistance (U.S. Pat. No. 6,228,992), bacterialdisease resistance (U.S. Pat. No. 5,516,671), plant growth anddevelopment (U.S. Pat. Nos. 6,723,897 and 6,518,488), starch production(U.S. Pat. Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295),modified oils production (U.S. Pat. Nos. 6,444,876; 6,426,447; and6,380,462), high oil production (U.S. Pat. Nos. 6,495,739; 5,608,149;6,483,008; and 6,476,295), modified fatty acid content (U.S. Pat. Nos.6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538;6,589,767; 6,537,750; 6,489,461; and 6,459,018), high protein production(U.S. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466),enhanced animal and human nutrition (U.S. Pat. Nos. 6,723,837;6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Pat.Nos. USRE37,543; 6,228,623; and U.S. Pat. Nos. 5,958,745, and6,946,588), environmental stress resistance (U.S. Pat. No. 6,072,103),pharmaceutical peptides and secretable peptides (U.S. Pat. Nos.6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processingtraits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No.6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzymeproduction (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No.6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seedproduction (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. Nos.6,576,818; 6,271,443; 5,981,834; and 5,869,720) and biofuel production(U.S. Pat. No. 5,998,700).

Alternatively, a gene of agronomic interest can affect the abovementioned plant characteristics or phenotypes by encoding a RNA moleculethat causes the targeted modulation of gene expression of an endogenousgene, for example by antisense (see, e.g. U.S. Pat. No. 5,107,065);inhibitory RNA (“RNAi,” including modulation of gene expression bymiRNA-, siRNA-, trans-acting siRNA-, and phased sRNA-mediatedmechanisms, e.g., as described in published applications U.S.2006/0200878 and U.S. 2008/0066206, and in U.S. patent application Ser.No. 11/974,469); or cosuppression-mediated mechanisms. The RNA couldalso be a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see,e.g., U.S. 2006/0200878) engineered to cleave a desired endogenous mRNAproduct. Methods are known in the art for constructing and introducingconstructs into a cell in such a manner that the transcribable DNAmolecule is transcribed into a molecule that is capable of causing genesuppression.

Selectable Markers

Selectable marker transgenes may also be used with the regulatoryelements of the invention. As used herein the term “selectable markertransgene” refers to any transcribable DNA molecule whose expression ina transgenic plant, tissue or cell, or lack thereof, can be screened foror scored in some way. Selectable marker genes, and their associatedselection and screening techniques, for use in the practice of theinvention are known in the art and include, but are not limited to,transcribable DNA molecules encoding β-glucuronidase (GUS), greenfluorescent protein (GFP), proteins that confer antibiotic resistance,and proteins that confer herbicide tolerance. An example of a selectablemarker transgene is provided as SEQ ID NO:42.

Cell Transformation

The invention is also directed to a method of producing transformedcells and plants that comprise one or more regulatory elements operablylinked to a transcribable DNA molecule.

The term “transformation” refers to the introduction of a DNA moleculeinto a recipient host. As used herein, the term “host” refers tobacteria, fungi, or plants, including any cells, tissues, organs, orprogeny of the bacteria, fungi, or plants. Plant tissues and cells ofparticular interest include protoplasts, calli, roots, tubers, seeds,stems, leaves, seedlings, embryos, and pollen.

As used herein, the term “transformed” refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is inherited by subsequent progeny. A“transgenic” or “transformed” cell or organism may also include progenyof the cell or organism and progeny produced from a breeding programemploying such a transgenic organism as a parent in a cross andexhibiting an altered phenotype resulting from the presence of a foreignDNA molecule. The introduced DNA molecule may also be transientlyintroduced into the recipient cell such that the introduced DNA moleculeis not inherited by subsequent progeny. The term “transgenic” refers toa bacterium, fungus, or plant containing one or more heterologous DNAmolecules.

There are many methods well known to those of skill in the art forintroducing DNA molecules into plant cells. The process generallycomprises the steps of selecting a suitable host cell, transforming thehost cell with a vector, and obtaining the transformed host cell.Methods and materials for transforming plant cells by introducing aplant construct into a plant genome in the practice of this inventioncan include any of the well-known and demonstrated methods. Suitablemethods include, but are not limited to, bacterial infection (e.g.,Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., byPEG-mediated transformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, and accelerationof DNA coated particles), and gene editing (e.g., CRISPR-Cas systems),among others.

This disclosure further contemplates that the disclosed syntheticexpression elements can be engineered in planta by using various geneediting methods known in the art. Such technologies used for genomeediting include, but are not limited to, ZFN (zinc-finger nuclease),meganucleases, TALEN (Transcription activator-like effector nucleases),and CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR-associated) systems. These genome editing methodscan be used to alter the expression element sequence within a plant cellto a different sequence.

Host cells may be any cell or organism, such as a plant cell, algalcell, algae, fungal cell, fungi, bacterial cell, or insect cell. Inspecific embodiments, the host cells and transformed cells may includecells from crop plants.

A transgenic plant subsequently may be regenerated from a transgenicplant cell of the invention. Using conventional breeding techniques orself-pollination, seed may be produced from this transgenic plant. Suchseed, and the resulting progeny plant grown from such seed, will containthe recombinant DNA molecule of the invention, and therefore will betransgenic.

Transgenic plants of the invention can be self-pollinated to provideseed for homozygous transgenic plants of the invention (homozygous forthe recombinant DNA molecule) or crossed with non-transgenic plants ordifferent transgenic plants to provide seed for heterozygous transgenicplants of the invention (heterozygous for the recombinant DNA molecule).Both such homozygous and heterozygous transgenic plants are referred toherein as “progeny plants.” Progeny plants are transgenic plantsdescended from the original transgenic plant and containing therecombinant DNA molecule of the invention. Seeds produced using atransgenic plant of the invention can be harvested and used to growgenerations of transgenic plants, i.e., progeny plants of the invention,comprising the construct of this invention and expressing a gene ofagronomic interest. Descriptions of breeding methods that are commonlyused for different crops can be found in one of several reference books,see, e.g., Allard, Principles of Plant Breeding, John Wiley & Sons, NY,U. of CA, Davis, Calif., 50-98 (1960); Simmonds, Principles of CropImprovement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen,Plant breeding Perspectives, Wageningen (ed), Center for AgriculturalPublishing and Documentation (1979); Fehr, Soybeans: Improvement,Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr,Principles of Variety Development, Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376 (1987).

The transformed plants may be analyzed for the presence of the gene orgenes of interest and the expression level and/or profile conferred bythe regulatory elements of the invention. Those of skill in the art areaware of the numerous methods available for the analysis of transformedplants. For example, methods for plant analysis include, but are notlimited to, Southern blots or northern blots, PCR-based approaches,biochemical analyses, phenotypic screening methods, field evaluations,and immunodiagnostic assays. The expression of a transcribable DNAmolecule can be measured using TaqMan® (Applied Biosystems, Foster City,Calif.) reagents and methods as described by the manufacturer and PCRcycle times determined using the TaqMan® Testing Matrix. Alternatively,the Invader® (Third Wave Technologies, Madison, Wis.) reagents andmethods as described by the manufacturer can be used to evaluatetransgene expression.

The invention also provides for parts of a plant of the invention. Plantparts include, but are not limited to, leaves, stems, roots, tubers,seeds, endosperm, ovule, and pollen. Plant parts of the invention may beviable, nonviable, regenerable, and/or non-regenerable. The inventionalso includes and provides transformed plant cells comprising a DNAmolecule of the invention. The transformed or transgenic plant cells ofthe invention include regenerable and/or non-regenerable plant cells.

The invention also provides a commodity product that is produced from atransgenic plant or part thereof containing the recombinant DNA moleculeof the invention. Commodity products of the invention contain adetectable amount of DNA comprising a DNA sequence selected from thegroup consisting of SEQ ID NOs:1-29 and SEQ ID NOs:43-45. As usedherein, a “commodity product” refers to any composition or product whichis comprised of material derived from a transgenic plant, seed, plantcell, or plant part containing the recombinant DNA molecule of theinvention. Commodity products include but are not limited to processedseeds, grains, plant parts, and meal. A commodity product of theinvention will contain a detectable amount of DNA corresponding to therecombinant DNA molecule of the invention. Detection of one or more ofthis DNA in a sample may be used for determining the content or thesource of the commodity product. Any standard method of detection forDNA molecules may be used, including methods of detection disclosedherein.

The invention may be more readily understood through reference to thefollowing examples, which are provided by way of illustration, and arenot intended to be limiting of the invention, unless specified. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the following examples represent techniques discovered bythe inventors to function well in the practice of the invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention, thereforeall matter set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

EXAMPLES Example 1 Design, Synthesis, and Cloning of SyntheticRegulatory Elements

The regulatory elements provided in Table 1 are novel syntheticexpression elements designed through algorithmic methods. Thesecomputationally-designed synthetic regulatory elements were chemicallysynthesized and cloned to make synthetic regulatory expression elementgroups (EXPs). Well over 1,000 synthetic regulatory elements weredesigned and assayed in soybean protoplasts and stably transformedsoybean plants to identify those synthetic regulatory elements thatprovided desired characteristics, such as protein expression levels andpatterns of expression. The synthetic regulatory elements described inTable 1 provide various patterns of expression useful in drivingexpression of many different coding sequences and interfering RNAs ofagronomic interest.

The computationally-designed synthetic regulatory elements do not haveextended homology to any known nucleic acid sequences that exist innature. The synthetic EXPs and the corresponding promoters, leaders,introns and 3′ UTRs are presented in Table 1. The synthetic EXPs werecloned using methods known in the art into binary plant transformationvectors, operably linked to a β-glucuronidase (GUS) coding sequence, andthe vectors were used to evaluate the levels and patterns of expressionprovided by the synthetic EXPs in stably transformed soybean, cotton andcorn plants.

Analysis of the synthetic regulatory element transcription start site(TSS) and intron/exon splice junctions can be performed usingtransformed plant tissue. Briefly, the plants are transformed with theplant expression vectors comprising the cloned DNA fragments operablylinked to a heterologous transcribable DNA molecule. Next, the 5′ RACESystem for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen,Carlsbad, Calif. 92008) is used to confirm the synthetic regulatoryelement TSS and intron/exon splice junctions by analyzing the DNAsequence of the produced mRNA transcripts.

TABLE 1 Synthetic transcriptional regulatory expression element groups,promoters, leaders, introns, and 3′ UTRs. Description and/or regulatoryelements SEQ ID Size of EXP linked in 5′→3′ direction Annotation NO:(bp) (SEQ ID NOs): EXP-At.GSP442.nno + 1 855 EXP: P-At.GSP442.nno:2 (SEQID NO: 2), At.Cyco:3 L-At.GSP442.nno:1 (SEQ ID NO: 3), I-At.Cyco:2 (SEQID NO: 33) P-At.GSP442.nno:2 2 480 Promoter L-At.GSP442.nno:1 3 20Leader EXP-At.GSP571 4 500 EXP: P-At.GSP571.nno:5 (SEQ ID NO: 5),L-At.GSP571.nno:1 (SEQ ID NO: 6) P-At.GSP571.nno:5 5 451 PromoterL-At.GSP571.nno:1 6 49 Leader EXP-At.GSP571.nno + 7 855 EXP:P-At.GSP571.nno:5 (SEQ ID NO: 5), At.Cyco:2 L-At.GSP571.nno:1 (SEQ IDNO: 6), I-At.Cyco:2 (SEQ ID NO: 33) EXP-At.GSP571.nno + 8 816 EXP:P-At.GSP571.nno:5 (SEQ ID NO: 5), At.GSI21.nno:10 L-At.GSP571.nno:1 (SEQID NO: 6), I-At.GSI21.nno:2 (SEQ ID NO: 9) I-At.GSI21.nno:2 9 309 IntronEXP-At.GSP571.nno + 10 810 EXP: P-At.GSP571.nno:5 (SEQ ID NO: 5),At.GSI102.nno:1 L-At.GSP571.nno:1 (SEQ ID NO: 6), I-At.GSI102.nno:1 (SEQID NO: 11) I-At.GSI102.nno:1 11 310 Intron EXP-At.GSP564 12 500 EXP:P-At.GSP564.nno:3 (SEQ ID NO: 13), L-At.GSP564.nno:1 (SEQ ID NO: 14)P-At.GSP564.nno:3 13 461 Promoter L-At.GSP564.nno:1 14 39 LeaderEXP-At.GSP564.nno + 15 855 EXP: P-At.GSP564.nno:3 (SEQ ID NO: 13),At.Cyco:2 L-At.GSP564.nno:1 (SEQ ID NO: 14), I-At.Cyco:2 (SEQ ID NO: 33)EXP-At.GSP564.nno + 16 807 EXP: P-At.GSP564.nno:3 (SEQ ID NO: 13),At.GSI17.nno:2 L-At.GSP564.nno:1 (SEQ ID NO: 14), I-At.GSI17.nno:1 (SEQID NO: 17) I-At.GSI17.nno:1 17 300 Intron EXP-At.GSP564.nno + 18 810EXP: P-At.GSP564.nno:3 (SEQ ID NO: 13), At.GSI102.nno:1L-At.GSP564.nno:1 (SEQ ID NO: 14), I-At.GSI102.nno:1 (SEQ ID NO: 11)EXP-At.GSP579 19 500 EXP: P-At.GSP579.nno:2 (SEQ ID NO: 20),L-At.GSP579.nno:1 (SEQ ID NO: 21) P-At.GSP579.nno:2 20 449 PromoterL-At.GSP579.nno:1 21 51 Leader EXP-At.GSP579.nno + 22 810 EXP:P-At.GSP579.nno:2 (SEQ ID NO: 20), At.GSI102.nno:3 L-At.GSP579.nno:1(SEQ ID NO: 21), I-At.GSI102.nno:1 (SEQ ID NO: 11) EXP-At.GSP571.nno +23 1350 EXP: E-At.GSP571.nno:1 (SEQ ID NO: 24), At.GSP442.nno +P-At.GSP442.nno:2 (SEQ ID NO: 2), At.Cyco:1 L-At.GSP442.nno:1 (SEQ IDNO: 3), L-At.Cyco-1:1:2 (SEQ ID NO: 40), I-At.Cyco:2 (SEQ ID NO: 33)E-At.GSP571.nno:1 24 422 Enhancer P-At.GSP571/442 25 902 ChimericPromoter: E-At.GSP571.nno:1 (SEQ ID NO: 24), P-At.GSP442.nno:2 (SEQ IDNO: 2) EXP-At.GSP576.nno + 26 800 EXP: P-At.GSP576.nno:4 (SEQ ID NO:27), At.GSI17.nno:3 L-At.GSP576.nno:2 (SEQ ID NO: 28), I-At.GSI17.nno:1(SEQ ID NO: 17) P-At.GSP576.nno:4 27 458 Promoter L-At.GSP576.nno:2 2842 Leader T-Zm.GST59.nno:1 29 400 3′ UTR EXP-At.GSP221 + 30 947 EXP:P-At.GSP221:3 (SEQ ID NO: 31), At.Cyco:3 L-At.GSP221:1 (SEQ ID NO: 32),I-At.Cyco:2 (SEQ ID NO: 33) P-At.GSP221:3 31 370 Promoter L-At.GSP221:132 229 Leader EXP-At.GSP442 + 43 928 EXP: P-At.GSP442.nno:2 (SEQ ID NO:2), L-I-At.Cyco L-At.GSP442.nno:1 (SEQ ID NO: 3), L-At.Cyco-1:1:2 (SEQID NO: 40), I-At.Cyco:2 (SEQ ID NO: 33) T-Zm.GST7.nno:2 44 300 3′ UTREXP-At.GSP576.nno + 45 855 EXP: P-At.GSP576.nno:4 (SEQ ID NO: 27),At.Cyco:1 L-At.GSP576.nno:2 (SEQ ID NO: 28), I-At.Cyco:2 (SEQ ID NO: 33)

Example 2 Analysis of the Synthetic EXPs, EXP-At.GSP442.Nno+At.Cyco:3and EXP-At.GSP221+At.Cyco:3, Driving GUS Expression in StablyTransformed Soybean Plants

Soybean plants were transformed with vectors, specifically plantexpression vectors containing regulatory element groups drivingexpression of the β-glucuronidase (GUS) transgene. The resulting plantswere analyzed for GUS protein expression to assess the effect of theselected regulatory element groups on expression.

Soybean plants were transformed with plant GUS expression constructscomprising the endogenous EXP, EXP-At.Cyco:1:1 (SEQ ID NO:38), and twosynthetic EXPs, EXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO:1) andEXP-At.GSP221+At.Cyco:3 (SEQ ID NO:30). EXP-At.Cyco:1:1 (SEQ ID NO:38)is derived from a Cytochrome c oxidase subunit VIa gene from Arabidopsisand is comprised of the promoter, P-At.Cyco-1:1:2 (SEQ ID NO:39),operably linked 5′ to the leader, L-At.Cyco-1:1:2 (SEQ ID NO:40), whichis operably linked 5′ to an intron, I-At.Cyco-1:1:1 (SEQ ID NO:41).EXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO:1) and EXP-At.GSP221+At.Cyco:3(SEQ ID NO:30) each comprised a synthetic promoter and leader operablylinked 5′ to the intron, I-At.Cyco:2 (SEQ ID NO:33). The sequence ofI-At.Cyco:2 (SEQ ID NO:33) is identical to the sequence ofI-At.Cyco-1:1:1 (SEQ ID NO:41), with the exception that there are twonucleotides after the intron splice site included in the sequence ofI-At.Cyco-1:1:1. Both I-At.Cyco introns splice the same.

The regulatory elements were cloned into base plant expression vectorsusing standard methods known in the art. The resulting plant expressionvectors contained a right border region from Agrobacterium tumefaciens(B-AGRtu.right border), a first transgene selection cassette used forselection of transformed plant cells that confers resistance to theantibiotic, spectinomycin; a second transgene cassette to assess theactivity of the regulatory element, which comprised an EXP sequenceoperably linked 5′ to a coding sequence for β-glucuronidase (GUS,GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO:42) containing a processable intronderived from the potato light-inducible tissue-specific ST-LS1 gene(Genbank Accession: X04753), operably linked 5′ to a 3′ UTR from theGossypium barbadense FbLate-2 gene (T-Gb.FbL2:1, SEQ ID NO:36); and aleft border region from Agrobacterium tumefaciens (B-AGRtu.left border).

Soybean plant cells were transformed by Agrobacterium-mediatedtransformation using these binary transformation vector constructs, asis well known in the art. The resulting transformed plant cells wereinduced to form whole soybean plants.

Histochemical GUS analysis was used for qualitative and quantitativeexpression analysis of the transformed plants. Whole tissue sectionswere incubated with GUS staining solution X-Gluc(5-bromo-4-chloro-3-indolyl-b-glucuronide) (1 milligram/milliliter) foran appropriate length of time, rinsed, and visually inspected for bluecoloration. GUS activity was qualitatively determined by direct visualinspection or inspection under a microscope using selected plant organsand tissues.

For quantitative analysis of GUS expression, total protein was extractedfrom selected tissues of transformed soybean plants. One microgram oftotal protein was used with the fluorogenic substrate4-methyleumbelliferyl-β-D-glucuronide (MUG) in a total reaction volumeof 50 microliters. The reaction product, 4-methlyumbelliferone (4-MU),is maximally fluorescent at high pH, where the hydroxyl group isionized. Addition of a basic solution of sodium carbonate simultaneouslystops the assay and adjusts the pH for quantifying the fluorescentproduct. Fluorescence was measured with excitation at 365 nm, emissionat 445 nm using a Fluoromax-3 with Micromax Reader, with slit width setat excitation 2 nm and emission 3 nm. Values are provided in units ofnmol GUS/hour/mg total protein.

The following tissues were sampled for GUS expression in the R₀generation; V5 stage root, leaf-sink, and source-leaf; R1 stage root,leaf-petiole, leaf-source, and flowers; R3 stage seed-immature and pod;R5 stage seed-cotyledon; and R8 stage seed-embryo and seed-cotyledon.Table 2 shows the mean quantitative GUS expression for each of thesampled tissues driven by the tested EXP regulatory element groupswherein “ND” indicates the expression in a particular tissue was notdetermined.

TABLE 2 Mean quantitative GUS expression in stably transformed soybeanplants driven by synthetic regulatory element groups and the endogenousEXP, EXP-At.Cyco:1:1. EXP-At.GSP442.nno + EXP-At.GSP221 + DevelopmentalEXP-At.Cyco:1:1 At.Cyco:3 At.Cyco:3 Stage Organ (SEQ ID NO: 38) (SEQ IDNO: 1) (SEQ ID NO: 30) V5 Root 151 399 928 Leaf-Sink 39 65 59Leaf-Source 52 109 100 R1 Root ND 616 1893 Leaf-Petiole 97 470 136Leaf-Source 46 177 240 Flowers 71 277 140 R3 Seed-Immature 64 477 ND Pod84 575 702 R5 Seed-Cotyledon 91 564 58 R8 Seed-Embryo 57 149 301Seed-Cotyledon 100 1118 414

As can be seen in Table 2, each of the synthetic regulatory elementgroups has a unique pattern of expression in the tissues sampledcompared to the endogenous EXP. For example, the synthetic At.GSP442promoter, P-At.GSP442.nno:2 (SEQ ID NO:2), and leader, L-At.GSP442.nno:1(SEQ ID NO:3), of EXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO:1) providesgreater levels of GUS expression in all of the assayed organs relativeto the endogenous EXP-At.Cyco:1:1 (SEQ ID NO:38), which comprises anidentical intron sequence. Analysis of the TSS demonstrated a consistentTSS. The intron was properly excised in the resulting mRNA as expected.Further, the synthetic At.GSP221 promoter, P-AT.GSP221:3 (SEQ ID NO:31),and leader, L-At.GSP221:1 (SEQ ID NO:32), of EXP-At.GSP221+At.Cyco:3(SEQ ID NO:30) also provides higher levels of constitutive expression inmost organs assayed relative to the endogenous EXP-At.Cyco:1:1, anddemonstrates a consistent TSS. However, the TSS ofEXP-At.GSP221+At.Cyco:3 was not located in the predicted location—therewere multiple potential TATA elements. This creates potential concernsfor multiple transcripts, which could produce multiple coding sequences.As such, EXP-At.GSP221+At.Cyco:3 was not considered acceptable for usein driving transgene expression in stably transformed dicot plants. Thisdemonstrates one of the complexities in designing synthetic expressionelements. Numerous synthetic elements were assayed in the developmentand identification of synthetic expression elements, but only a smallsubset provided desirable characteristics and regulatory activity,illustrating the complexity in designing effective synthetictranscriptional regulatory elements.

As can be seen in Table 2, the synthetic promoter, P-At.GSP442.nno:2(SEQ ID NO:2) and L-At.GSP442.nno:1 (SEQ ID NO:3) comprised withinEXP-At.GSP442.nno+At.Cyco:3 (SEQ ID NO: 1) is able to drive constitutivetransgene expression of an operably linked transgene in a stablytransformed soybean plant.

Example 3 Analysis of the Synthetic At.GSP571 Promoter and Leader, andthe Synthetic At.GSI21 and At.GSI102 Introns, Driving GUS Expression inStably Transformed Soybean Plants

Soybean plants were transformed with vectors, specifically plantexpression vectors containing regulatory element groups drivingexpression of the β-glucuronidase (GUS) transgene. The resulting plantswere analyzed for GUS protein expression to assess the effect of theselected regulatory element groups on expression.

Soybean plants were transformed with plant GUS expression constructs,comprising the synthetic EXPs, EXP-At.GSP571 (SEQ ID NO:4),EXP-At.GSP571.nno+At.Cyco:2 (SEQ ID NO:7),EXP-At.GSP571.nno+At.GSI21.nno:10 (SEQ ID NO:8), andEXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO:10). Each of the syntheticEXPs comprised the synthetic At.GSP571 promoter (SEQ ID NO:5) and leader(SEQ ID NO:6). EXP-At.GSP571.nno+At.Cyco:2 comprised the endogenousArabidopsis intron, I-At.Cyco:2 (SEQ ID NO:33).EXP-At.GSP571.nno+At.GSI21.nno:10 and EXP-At.GSP571.nno+At.GSI102.nno:1comprised the synthetic introns, I-At.GSI21.nno:2 (SEQ ID NO:9) andI-At.GSI102.nno:1 (SEQ ID NO:11), respectively. The binary planttransformation vectors were similar to those described in Example 2 withthe exception that each of the At.GSP571 EXP vectors comprised the 3′UTR, T-Mt.Sali3-2-1:2:1 (SEQ ID NO:34), derived from the Sali3 gene ofMedicago truncatula.

Quantitative and qualitative GUS expression analysis was performed asdescribed in Example 2. Tissue samples used for analysis were the sameas that described in Example 2. Table 3 shows the mean quantitative GUSexpression for each of the sampled tissues driven by the testedsynthetic EXP regulatory elements, wherein “ND” indicates the expressionin a particular tissue was not determined.

TABLE 3 Mean quantitative GUS expression in stably transformed soybeanplants driven by synthetic regulatory elements. EXP-At.GSP571.nno +EXP-At.GSP571.nno + EXP-At.GSP571.nno + Developmental EXP-At.GSP571At.Cyco:2 At.GSI21.nno:10 At.GSI102.nno:1 Stage Organ (SEQ ID NO: 4)(SEQ ID NO: 7) (SEQ ID NO: 8) (SEQ ID NO: 10) V5 Root 40 57 165 579Leaf-Sink 650 612 792 1683 Leaf-Source 1379 1090 1475 2128 R1 Root 110ND 457 645 Leaf-Petiole 951 1091 1267 1167 Leaf-Source 1995 3538 20942129 Flowers 703 830 1408 350 R3 Seed-Immature 75 609 495 232 Pod 8522228 4014 1535 R5 Seed-Cotyledon 650 474 540 1433 R8 Seed-Embryo 11531004 603 1122 Seed-Cotyledon 2449 4524 2533 2648

As can be seen in Table 3, the synthetic At.GSP571 promoter and leaderprovide constitutive expression in all the organs assayed. Expressionwas highest in the leaf and seeds. Analysis of the TSS demonstrated aconsistent TSS. Operably linking an intron sequence altered expressionin many of the organs, providing a means to “fine-tune” the constitutiveexpression. Differences in expression were observed when operablylinking the synthetic introns, I-At.GSI21.nno:2 (SEQ ID NO:9) andI-At.GSI102.nno:1 (SEQ ID NO:11). The synthetic introns enhancedexpression in some tissues, but differed in the level of enhancement foreach organ. For example, enhancement using the synthetic intronI-At.GSI21.nno:2 in R3 pod was higher than the enhancement seen usingthe synthetic intron I-At.GSI102.nno:1 and the endogenous intronI-At.Cyco:2 relative to EXP-At.GSP571. Expression was only slightlyenhanced by the three operably linked introns in R1 petiole. In R1flowers, I-At.GSI21.nno:2 and I-At.Cyco:2 enhanced expression, withI-At.GSI21.nno:2 providing a high level of expression enhancement andI-At.Cyco:2 providing a moderate level of enhancement. Interestingly,I-At.GSI102.nno:1 reduced expression in R1 flowers.

Analysis of the resulting mRNAs showed proper and consistent processingof the intron elements.

The synthetic promoter, P-At.GSP571.nno:5 (SEQ ID NO:5) and leaderL-At.GSP571.nno:1 (SEQ ID NO:6) comprised within EXP-At.GSP571 (SEQ IDNO:4) provide constitutive expression of an operably linked transgene instably transformed soybean plants. The synthetic EXPs,EXP-At.GSP571.nno+At.Cyco:2 (SEQ ID NO:7), which comprises theArabidopsis intron I-At.Cyco:2 (SEQ ID NO:33), andEXP-At.GSP571.nno+At.GSI21.nno:10 (SEQ ID NO:8) andEXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO:10), which comprise thesynthetic introns I-At.GSI21.nno:2 (SEQ ID NO:9) and I-At.GSI102.nno:1(SEQ ID NO:11), respectively, provide unique patterns of constitutiveexpression in stably transformed soybean plants. The synthetic introns,I-At.GSI21.nno:2 (SEQ ID NO:9) and I-At.GSI102.nno:1 (SEQ ID NO: 11),provide enhanced or modulated expression in many of the plant organswhen operably linked to EXP-At.GSP571 (SEQ ID NO:4). These uniqueexpression patterns can be used to drive specific transgenes in whichthe specific expression pattern of one of the four At.GSP571 EXPs ismost desirable.

Example 4 Analysis of the Synthetic At.GSP564 Promoter and Leader, andthe Synthetic At.GSI17 and At.GSI102 Introns, Driving GUS Expression inStably Transformed Soybean Plants

Soybean plants were transformed with vectors, specifically plantexpression vectors containing regulatory element groups drivingexpression of the β-glucuronidase (GUS) transgene. The resulting plantswere analyzed for GUS protein expression to assess the effect of theselected regulatory element groups on expression.

Soybean plants were transformed with plant GUS expression constructs,comprising the synthetic EXPs, EXP-At.GSP564 (SEQ ID NO:12),EXP-At.GSP564.nno+At.Cyco:2 (SEQ ID NO:15),EXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO:16), andEXP-At.GSP564.nno+At.GSI102.nno:1 (SEQ ID NO:18). Each of the syntheticEXPs comprised the synthetic P-At.GSP564.nno:3 promoter (SEQ ID NO:13)and synthetic L-At.GSP564.nno.1 leader (SEQ ID NO:14).EXP-At.GSP564.nno+At.Cyco:2 comprised the Arabidopsis intron,I-At.Cyco:2 (SEQ ID NO:33). EXP-At.GSP564.nno+At.GSI17.nno:2 andEXP-At.GSP564.nno+At.GSI102.nno:1 comprised the synthetic introns,I-At.GSI17.nno:1 (SEQ ID NO:17) and I-At.GSI102.nno:1 (SEQ ID NO:11),respectively. The binary plant transformation vectors were similar tothose described in Example 2, with the exception that each of theAt.GSP564 EXP vectors comprised the 3′ UTR, T-Mt.Oxr-1:2:1 (SEQ IDNO:35), derived from a putative oxidoreductase (OXR) protein gene fromMedicago truncatula.

Quantitative and qualitative GUS expression analysis was performed asdescribed in Example 2. Tissue samples used for analysis were the sameas that described in Example 2. Table 4 shows the mean quantitative GUSexpression for each of the sampled tissues driven by the testedsynthetic EXP regulatory elements, wherein “ND” indicates the expressionin a particular tissue was not determined.

TABLE 4 Mean quantitative GUS expression in stably transformed soybeanplants driven by synthetic regulatory elements. EXP-At.GSP564.nno +EXP-At.GSP564.nno + EXP-At.GSP564.nno + Developmental EXP-At.GSP564At.Cyco:2 At.GSI17.nno:2 At.GSI102.nno:1 Stage Organ (SEQ ID NO: 12)(SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 18) V5 Root 61 108 54 145Leaf-Sink 38 220 89 259 Leaf-Source 74 421 209 1229 R1 Root 118 165 2348627 Leaf-Petiole 90 235 273 148 Leaf-Source 140 205 436 917 Flowers 6691 ND 305 R3 Seed-Immature 26 ND 101 ND Pod 40 ND 749 ND R5Seed-Cotyledon 25 88 78 61 R8 Seed-Embryo 38 97 137 70 Seed-Cotyledon 79288 655 572

As can be seen in Table 4, the synthetic At.GSP564 promoter and leaderprovide constitutive expression in all the organs assayed. Expressionwas highest in the leaf and seeds. Analysis of the TSS demonstrated aconsistent TSS. Operably linking an intron sequence altered expressionin many of the organs, providing a means to “fine-tune” the constitutiveexpression. Differences in expression were observed when operablylinking the synthetic introns, I-At.GSI17.nno:1 (SEQ ID NO:17) andI-At.GSI102.nno:1 (SEQ ID NO:11). The synthetic introns enhancedexpression in some tissues relative to EXP-At.GSP564, but differed inthe level of enhancement for each organ. For example, enhancement usingthe synthetic intron I-At.GSI102.nno:1 in V5 source leaf was higher thanthe enhancement seen using the synthetic intron I-At.GSI17.nno:1. In R1root, enhancement using the synthetic intron I-At.GSI17.nno:1 was higherthan the enhancement conferred by the synthetic intronI-At.GSI102.nno:1. Both synthetic introns provided greater enhancementof expression in R1 source leaf than the endogenous intron, I-At.Cyco:2.

Analysis of the resulting mRNAs showed proper and consistent processingof the intron elements.

The synthetic At.GSP564 promoter, P-At.GSP564.nno.3 (SEQ ID NO:13) andleader, L-At.GSP564.nno:1 (SEQ ID NO:14) comprising EXP-At.GSP564 (SEQID NO:12) provide constitutive expression of an operably linkedtransgene in stably transformed soybean plants. The synthetic EXPs,EXP-At.GSP564.nno+At.Cyco:2 (SEQ ID NO:15), which comprises theArabidopsis intron I-At.Cyco:2 (SEQ ID NO:33), andEXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO:16) andEXP-At.GSP564.nno+At.GSI102.nno:1 (SEQ ID NO:18), which comprise thesynthetic introns, I-At.GSI17.nno:1 (SEQ ID NO:17) and I-At.GSI102.nno:1(SEQ ID NO:11), respectively, provide unique patterns of constitutiveexpression in stably transformed soybean plants. The synthetic introns,I-At.GSI17.nno:1 (SEQ ID NO:17) and I-At.GSI102.nno:1 (SEQ ID NO:11),provide enhanced or modulated transgene expression in many of the plantorgans when operably linked to EXP-At.GSP564 (SEQ ID NO:12). Theseunique expression patterns can be used to drive specific transgenes inwhich the specific expression pattern of one of the four At.GS564 EXPsis most desirable.

Example 5 Analysis of the Synthetic EXP,EXP-At.GSP579.Nno+At.GSI102.Nno:3, Driving GUS Expression in StablyTransformed Soybean Plants

Soybean plants were transformed with vectors, specifically plantexpression vectors containing a synthetic regulatory element groupdriving expression of the 1-glucuronidase (GUS) transgene. The resultingplants were analyzed for GUS protein expression to assess the effect ofthe selected synthetic regulatory element group on expression.

Soybean plants were transformed with a plant GUS expression construct,comprising the synthetic EXP, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ IDNO:22). EXP-At.GSP579.nno+At.GSI102.nno:3 comprises EXP-At.GSP579 (SEQID NO:19) consisting of the At.GSP promoter and leader (SEQ ID NOs:20and 21, respectively), operably linked 5′ to the synthetic intron,I-At.GSI102.nno:1 (SEQ ID NO:11). The GUS transgene cassette alsocomprises the 3′ UTR, T-Mt.RD22-1:2:1 (SEQ ID NO:37) derived from adehydration-responsive protein RD22 gene from Medicago truncatula.

Quantitative and qualitative GUS expression analysis was performed asdescribed in Example 2. Tissue samples used for analysis were the sameas that described in Example 2. Table 5 shows the mean quantitative GUSexpression for each of the sampled tissues driven by the synthetic EXP,EXP-At.GSP579.nno+At.GSI102.nno:3, wherein “ND” indicates the expressionin a particular tissue was not determined.

TABLE 5 Mean quantitative GUS expression in stably transformed soybeanplants driven by EXP-At.GSP579.nno + At.GSI102.nno:3.EXP-At.GSP579.nno + Developmental At.GSI102.nno:3 Stage Organ (SEQ IDNO: 22) V5 Root 187 Leaf-Sink 311 Leaf-Source 458 R1 Root 148Leaf-Petiole 118 Leaf-Source 425 Flowers 130 R3 Seed-Immature ND Pod NDR5 Seed-Cotyledon ND R8 Seed-Embryo 127 Seed-Cotyledon 266

As can be seen in Table 5, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ IDNO:22) provides constitutive expression in stably transformed soybeanplants. The synthetic promoter P-At.GSP579.nno:2 (SEQ ID NO:20) andleader L-At.GSP579.nno:1 (SEQ ID NO:21) comprised within EXP-At.GSP579(SEQ ID NO: 19) drive constitutive expression of an operably linkedtransgene. It can be inferred by the previous Examples in which thesynthetic intron, I-At.GSI102.nno:1 (SEQ ID NO: 11), was operably linkedto other constitutive synthetic promoters that I-At.GSI102.nno:1enhanced or modulated the constitutive expression imparted byEXP-At.GSP579 in at least some of the organs sampled.

Example 6 Analysis of the Synthetic EXP, EXP-At.GSP571.Nno+At.Cyco:2,Driving GUS Expression in Stably Transformed Cotton Plants

Cotton plants were transformed with a vector, specifically a plantexpression vector containing a synthetic regulatory element groupdriving expression of the 1-glucuronidase (GUS) transgene. The resultingplants were analyzed for GUS protein expression to assess the effect ofthe synthetic regulatory element group on expression.

A plant binary vector comprising the synthetic EXP,EXP-At.GSP571.nno+At.Cyco:2 (SEQ ID NO:7), similar to that described inExample 3, was used to stably transform cotton plants. The GUS transgenecassette comprised EXP-At.GSP571.nno+At.Cyco:2 operably linked 5′ to acoding sequence for 1-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ IDNO:42) containing a processable intron derived from the potatolight-inducible tissue-specific ST-LS1 gene (Genbank Accession: X04753),operably linked 5′ to a 3′ UTR from the Gossypium barbadense FbLate-2gene (T-Gb.FbL2:1, SEQ ID NO:36). The resulting transformed cottonevents were grown and tissue samples were derived from 4Node Leaf; 8NodePetiole, Sink Leaf, and Source Leaf; Pre-fertilization Square Bracts andSquare Bud; Flowering Anther and Flower Ovary; and 8 Days AfterPollination (DAP) Boll Wall were sampled and assayed for qualitative andquantitative GUS expression.

Table 6 shows the mean quantitative GUS expression for each of thesampled tissues driven by the synthetic EXP,EXP-At.GSP571.nno+At.Cyco:2.

TABLE 6 Mean quantitative GUS expression in stably transformed cottonplants driven by EXP-At.GSP571.nno + At.Cyco:2. Stage Organ Mean 4NodeLeaf 1232.57 8Node Leaf, Petiole 223.68 Leaf, Sink 612.14 Leaf, Source618.9 Pre-fertilization Square Bracts 381.69 Square Bud 347.22 FloweringAnther 64.66 Flower, Ovary 210.92 8DAP Boll Wall 835.94

As can be seen in Table 6, EXP-At.GSP571.nno+At.Cyco:2 expressed in allthe tissues sampled. Expression was highest in 4Node Leaf and lowest inthe Flowering Anther.

Expression in 8Node Sink and Source Leaf were relatively the same andabout half that of the 4Node Leaf. Expression was also high in the BollWall. Table 6 demonstrates that the promoter, P-At.GSP571.nno:5 (SEQ IDNO:5), is able to drive constitutive expression in stably transformedcotton plants. The intron, I-At.Cyco:2 (SEQ ID NO:33), withinEXP-At.GSP571.nno+At.Cyco:2 enhanced expression of the P-At.GSP571.nno:5promoter in stably transformed soybean plants, as shown in Example 3.

Example 7 Analysis of the Synthetic Chimeric Promoter P-At.GSP571/442Driving GUS Expression in Stably Transformed Soybean Plants

Soybean plants were transformed with vectors, specifically plantexpression vectors containing regulatory element groups drivingexpression of the β-glucuronidase (GUS) transgene. The resulting plantswere analyzed for GUS protein expression to assess the effect of theselected synthetic regulatory element groups on expression.

Soybean plants were transformed with a plant binary vector comprisingthe synthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 (SEQ IDNO:23), which is comprised of the synthetic chimeric promoterP-At.GSP571/442 (SEQ ID NO:25) comprising a synthetic enhancerE-At.GSP571.nno:1 (SEQ ID NO:24) derived from the synthetic promoterP-At.GSP571.nno:5 (SEQ ID NO:5) which is operably linked 5′ to thesynthetic promoter P-At.GSP442.nno:2 (SEQ ID NO:2) and is operablylinked 5′ with the synthetic leader, L-At.GSP442.nno:1 (SEQ ID NO:3),operably linked 5′ to the leader, L-At.Cyco-1:1:2 (SEQ ID NO:40), whichis operably linked 5′ to the intron, I-At.Cyco:2 (SEQ ID NO:33). The GUStransgene cassette comprised EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1operably linked 5′ to a coding sequence for β-glucuronidase (GUS,GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO:42) containing a processable intronderived from the potato light-inducible tissue-specific ST-LS1 gene(Genbank Accession: X04753), operably linked 5′ to the synthetic 3′ UTR,T-Zm.GST59.nno:1 (SEQ ID NO:29).

A plant binary vector used to compare the activity of the chimericpromoter was also constructed. The vector comprised an EXP,EXP-At.GSP442+L-I-At.Cyco (SEQ ID NO:43), which is comprised of thesynthetic promoter, P-At.GSP442.nno:2 (SEQ ID NO:2), operably linked 5′to the synthetic leader, L-At.GSP442.nno:1 (SEQ ID NO:3), operablylinked 5′ to the leader, L-At.Cyco-1:1:2 (SEQ ID NO:40), which isoperably linked 5′ to the intron, I-At.Cyco:2 (SEQ ID NO:33). The binaryvectors are similar to those described in Examples 2-6, with theexception that each GUS transgene cassette has the synthetic 3′ UTR,T-Zm.GST59.nno:1 (SEQ ID NO:29) operably linked 3′ to the GUS codingsequence.

Soybean plants were transformed with the two binary vectors. Tissuesamples were taken of selected organs at specific developmental stagesand assayed for qualitative and quantitative GUS expression. Table 7shows the mean quantitative GUS expression for each of the sampledtissues driven by the synthetic EXPs,EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 and EXP-At.GSP442+L-I-At.Cyco.

TABLE 7 Mean quantitative GUS expression in stably transformed soybeanplants driven by EXP-At.GSP571.nno + At.GSP442.nno + At.Cyco:1 andEXP-At.GSP442 + L-I-At.Cyco. EXP-At.GSP571.nno + EXP-At.GSP442 +At.GSP442.nno + L-I-At.Cyco At.Cyco:1 (SEQ ID NO: 43) (SEQ ID NO: 23)Stage Organ Mean Mean V5 Leaf, Sink 69.61 72.12 Leaf, 88.22 96.06 SourceRoot 74.67 102.9 R1 Flowers 79.16 62.01 Leaf, 77.07 87 Petiole Leaf,66.59 114.33 Source Root 76.88 123.12 R3 Pod 93.19 102.54 Seed, 71.1561.62 Immature R5 Seed, 78.72 92.83 Cotyledon R8 Seed, 65.55 72.15Cotyledon Seed, 129.95 107.66 Embryo

As can be seen in Table 7, the addition of the synthetic enhancerE-At.GSP571.nno:1 enhanced expression in many of the tissues sampled.Both EXPs provided constitutive expression in the stably transformedsoybean plants. The synthetic 3′ UTR, T-Zm.GST59.nno:1, functioned in asimilar manner as a native 3′ UTR in providing proper termination andpolyadenylation of the transcript.

Example 8 Analysis of the Synthetic Chimeric Promoter P-At.GSP571/442Driving GUS Expression in Stably Transformed Cotton Plants

Cotton plants were transformed with a vector, specifically a plantexpression vector containing a synthetic regulatory element groupdriving expression of the β-glucuronidase (GUS) transgene. The resultingplants were analyzed for GUS protein expression to assess the effect ofthe selected synthetic regulatory element group on expression.

Cotton plants were transformed with a plant binary vector comprising thesynthetic EXP, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 (SEQ ID NO:23),which is comprised of the synthetic chimeric promoter P-At.GSP571/442(SEQ ID NO:25) comprising a synthetic enhancer E-At.GSP571.nno:1 (SEQ IDNO:24) derived from the synthetic promoter P-At.GSP571.nno:5 (SEQ IDNO:5) which is operably linked 5′ to the synthetic promoterP-At.GSP442.nno:2 (SEQ ID NO:2) and is operably linked 5′ to thesynthetic leader, L-At.GSP442.nno:1 (SEQ ID NO:3), operably linked 5′ tothe leader, L-At.Cyco-1:1:2 (SEQ ID NO:40), which is operably linked 5′to the intron, I-At.Cyco:2 (SEQ ID NO:33). The GUS transgene cassettecomprised EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 operably linked 5′to a coding sequence for β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1:1:1,SEQ ID NO:42) containing a processable intron derived from the potatolight-inducible tissue-specific ST-LS1 gene (Genbank Accession: X04753),operably linked 5′ to the synthetic 3′ UTR, T-Zm.GST59.nno:1 (SEQ IDNO:29). The resulting transformed cotton events were grown and tissuesamples derived from 4Node Leaf; 8Node Petiole, Sink Leaf, and SourceLeaf; Pre-fertilization Square Bracts and Square Bud; Flowering Antherand Flower Ovary; and 8 Days After Pollination (DAP) Boll Wall weresampled and assayed for qualitative and quantitative GUS expression.

Table 8 shows the mean quantitative GUS expression for each of thesampled tissues driven by the synthetic EXP,EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1 wherein “bdl” means belowdetection limit.

TABLE 8 Mean quantitative GUS expression in stably transformed cottonplants driven by EXP-At.GSP571.nno + At.GSP442.nno + At.Cyco:1. StageOrgan Mean 4Node Leaf 177.74 8Node Leaf, Petiole bdl Leaf, Sink 108.39Leaf, Source 294.99 Pre-fertilization Square Bracts 78.84 Square Bud118.21 Flowering Anther 69.19 Flower, Ovary 69.78 8DAP Boll Wall 159.58

As can be seen in Table 8, EXP-At.GSP571.nno+At.GSP442.nno+At.Cyco:1(SEQ ID NO:23) was able to drive constitutive GUS expression in thetissues sampled. Expression in the Petiole was determined to be belowdetection limits. Expression was highest in 8Node Source Leaf.Expression was relatively equal in the Flowering Anther and FlowerOvary. In addition, the synthetic 3′ UTR, T-Zm.GST59.nno:1 (SEQ IDNO:29) functioned in a similar manner as a native 3′ UTR in providingproper termination and polyadenylation of the transcript.

Example 9 Analysis of the Synthetic EXP, EXP-At.GSP576.Nno+At.Cyco:1,Driving GUS Expression in Stably Transformed Soybean Plants

Soybean plants were transformed with a vector, specifically a plantexpression vector containing a synthetic regulatory element groupdriving expression of the 1-glucuronidase (GUS) transgene. The resultingplants were analyzed for GUS protein expression to assess the effect ofthe selected synthetic regulatory element group on expression.

Soybean plants were transformed with a plant binary vector comprisingthe synthetic EXP, EXP-At.GSP576.nno+At.Cyco:1 (SEQ ID NO:45). The GUStransgene cassette also comprised the 3′ UTR from the Gossypiumbarbadense FbLate-2 gene (T-Gb.FbL2:1, SEQ ID NO:36), operably linked 3′to the GUS coding sequence. The resulting transformed soybean eventswere grown and tissue samples of selected organs from severaldevelopmental stages were sampled and assayed for qualitative andquantitative GUS expression. Expression of GUS in the stably transformedsoybean plants, driven by EXP-At.GSP576.nno+At.Cyco:1, is presented inTable 9.

TABLE 9 Mean quantitative GUS expression in stably transformed soybeanplants driven by EXP-At.GSP576.nno + At.Cyco:1. Developmental StageOrgan Mean V5 Root 60.95 Leaf-Sink 97.43 Leaf-Source 181.64 R1 Root 82.4Leaf-Petiole 208.28 Leaf-Source 214 Flowers 123.37 R3 Seed-Immature95.29 Pod 158.24 R5 Seed-Cotyledon 85.97 R8 Seed-Embryo 67.4Seed-Cotyledon 52.92

As can be seen in Table 9, EXP-At.GSP576.nno+At.Cyco:1 (SEQ ID NO:45)provided constitutive expression in stably transformed soybean plants.The synthetic promoter P-At.GSP576.nno:4 (SEQ ID NO:27) and leaderL-At.GSP576.nno:2 (SEQ ID NO:28) drive constitutive expression of anoperably linked transgene. It can be inferred by the previous Examplesin which the intron, I-At.Cyco:2 (SEQ ID NO:33), was operably linked toother constitutive synthetic promoters, that I-At.Cyco:2 enhanced ormodulated the constitutive expression imparted by P-At.GSP576.nno:4 inat least some of the organs sampled.

Example 10 Analysis of the Synthetic EXP,EXP-At.GSP576.Nno+At.GSI17.Nno:3, Driving GUS Expression in StablyTransformed Soybean Plants

Soybean plants are transformed with vectors, specifically plantexpression vectors containing regulatory element groups drivingexpression of the 1-glucuronidase (GUS) transgene. The resulting plantsare analyzed for GUS protein expression to assess the effect of theselected regulatory element groups on expression.

Soybean plants are transformed with plant binary vectors comprisingeither the synthetic EXP, EXP-At.GSP576.nno+At.GSI17.nno:3 (SEQ IDNO:26), or the EXP, EXP-At.Cyco:1:1 (SEQ ID NO:38). The GUS transgenecassettes also comprise the 3′ UTR from the Gossypium barbadenseFbLate-2 gene (T-Gb.FbL2:1, SEQ ID NO:36) operably linked 3′ to the GUScoding sequence. The resulting transformed soybean events are grown andtissue samples of selected organs from several developmental stages aresampled and assayed for qualitative and quantitative GUS expression.Expression of GUS in the stably transformed soybean plants, driven byEXP-At.GSP576.nno+At.GSI17.nno:3, is compared to the expression drivenby EXP-At.Cyco:1:1. Expression of GUS in stably transformed soybeanplants driven by EXP-At.GSP576.nno+At.GSI17.nno:3 is demonstrative ofthe ability of the synthetic promoter P-At.GSP576.nno:4 (SEQ ID NO:27)and leader L-At.GSP576.nno:2 (SEQ ID NO:28) to drive constitutiveexpression of an operably linked transgene.

As demonstrated in Examples 9 and 11, the synthetic promoterP-At.GSP576.nno:4 (SEQ ID NO:27) and leader L-At.GSP576.nno:2 (SEQ IDNO:28) drive constitutive expression of an operably linked transgene. Aswas demonstrated in Example 4, the synthetic intron, I-At.GSI17.nno:1(SEQ ID NO:17) enhanced or modulated transgene expression in many of theplant organs when operably linked to EXP-At.GSP564 (SEQ ID NO:12).Likewise, it can be reasonably expected that expression of the syntheticpromoter P-At.GSP576.nno:4 and leader L-At.GSP576.nno:2 would beenhanced or modulated in a similar manner.

Example 11 Analysis of the Synthetic EXP,EXP-At.GSP576.Nno+At.GSI17.Nno:3, Driving GUS Expression in StablyTransformed Cotton Plants

Cotton plants were transformed with a vector, specifically a plantexpression vector containing a synthetic regulatory element groupdriving expression of the β-glucuronidase (GUS) transgene. The resultingplants were analyzed for GUS protein expression to assess the effect ofthe selected synthetic regulatory element group on expression.

Cotton plants were transformed with a binary vector comprising thesynthetic EXP, EXP-At.GSP576.nno+At.GSI17.nno:3 (SEQ ID NO:26), aspreviously described in Example 10. The GUS transgene cassettes alsocomprised the 3′ UTR from the Gossypium barbadense FbLate-2 gene(T-Gb.FbL2:1, SEQ ID NO:36) operably linked 3′ to the GUS codingsequence. The resulting transformed cotton events were grown and tissuesamples derived from 4Node Leaf; 8Node Petiole, Sink Leaf, and SourceLeaf; Pre-fertilization Square Bracts and Square Bud; Flowering Antherand Flower Ovary; and 8 Days After Pollination (DAP) Boll Wall weresampled and assayed for qualitative and quantitative GUS expression.

Table 10 shows the mean quantitative GUS expression for each of thesampled tissues driven by the syntheticEXP-At.GSP576.nno+At.GSI17.nno:3.

TABLE 10 Mean quantitative GUS expression in stably transformed cottonplants driven by EXP-At.GSP576.nno + At.GSI17.nno:3. Stage Organ Mean4Node Leaf 579.03 8Node Leaf, Petiole 301.57 Leaf, Sink 159.4 Leaf,Source 577.11 Pre-fertilization Square Bracts 262.66 Square Bud 223.59Flowering Anther 171.2 Flower, Ovary 109 8DAP Boll Wall 433.64

As can be seen in Table 10, EXP-At.GSP576.nno+At.GSI17.nno:3 (SEQ ID NO:26) drove constitutive expression of the GUS transgene in stablytransformed cotton plants. Expression was highest in 4Node Leaf, 8NodeSource Leaf, and 8DAP Boll Wall. The synthetic promoterP-At.GSP576.nno:4 (SEQ ID NO:27) and leader L-At.GSP576.nno:2 (SEQ IDNO:28) are able to drive constitutive expression of an operably linkedtransgene in stably transformed cotton plants. As was demonstrated inExample 4, the synthetic intron, I-At.GSI17.nno:1 (SEQ ID NO:17),enhanced or modulated transgene expression in many of the plant organswhen operably linked to EXP-At.GSP564 (SEQ ID NO: 12). Likewise, it canbe reasonably expected that expression of the synthetic promoter,P-At.GSP576.nno:4 and leader, L-At.GSP576.nno:2, would be enhanced ormodulated in a similar manner in stably transformed cotton plants.

Example 12 Enhancer Elements Derived from the Regulatory Element

Enhancers are derived from the promoter elements presented as SEQ IDNOs: 2, 5, 13, 20, 25, 27, 31, and 39. The enhancer element may becomprised of one or more cis regulatory elements that when operablylinked 5′ or 3′ to a promoter element, or operably linked 5′ or 3′ toadditional enhancer elements that are operably linked to a promoter, canenhance or modulate expression levels of a transcribable DNA molecule,or provide expression of a transcribable DNA molecule in a specific celltype or plant organ or at a particular time point in development orcircadian rhythm. Enhancers are made by removing the TATA box orfunctionally similar elements and any downstream sequence from thepromoters that allow transcription to be initiated from the promoterspresented as SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31, and 39 or fragmentsthereof. For example, the synthetic enhancer, E-At.GSP571.nno:1 (SEQ IDNO:24) was derived from the synthetic promoter, P-At.GSP571.nno:5 (SEQID NO:5) and consists of nucleotides 1 through 422 of P-At.GSP571.nno:5,eliminating the 3′ downstream sequence which also contains the TATA boxof the synthetic promoter.

Further refinement of the enhancer element may be required and isvalidated empirically. In addition, position of the enhancer elementrelative to other elements within a chimeric regulatory element group isalso empirically determined, since the order of each element within thechimeric regulatory element group may impart different effects,depending upon the relative positions of each element. Some promoterelements will have multiple TATA box or TATA box-like elements andpotentially multiple transcription start sites. Under thosecircumstances, it may be necessary to first identify where the first TSSis located and then begin designing enhancers using the first TSS toprevent the potential initiation of transcription from occurring withina putative enhancer element.

Enhancer elements, derived from the synthetic promoter elementspresented as SEQ ID NOs: 2, 5, 13, 20, 25, 27, 31, and 39 are clonedusing methods known in the art to be operably linked 5′ or 3′ to apromoter element, or operably linked 5′ or 3′ to additional enhancerelements that are operably linked to a promoter. Alternatively, enhancerelements can be cloned, using methods known in the art, to provide alarger enhancer element that is comprised of two or more copies of theenhancer and cloned using methods known in the art to be operably linked5′ or 3′ to a promoter element, or operably linked 5′ or 3′ toadditional enhancer elements that are operably linked to a promoterproducing a chimeric transcriptional regulatory element. Enhancerelements can also be cloned using methods known in the art to beoperably linked 5′ to a promoter element derived from a different genusorganism, or operably linked 5′ or 3′ to additional enhancer elementsderived from other genus organisms that are operably linked to apromoter derived from either the same or different genus organism,resulting in a chimeric regulatory element. A GUS expression planttransformation vector may be constructed using methods known in the artsimilar to the constructs described in Example 2 in which the resultingplant expression vectors contain a right border region fromAgrobacterium tumefaciens (B-AGRtu.right border), a first transgeneselection cassette used for selection of transformed plant cells thatconfers resistance to the antibiotic, spectinomycin; and a secondtransgene cassette to test the enhancer element comprised of, theenhancer element operably linked 5′ or 3′ to a promoter element oroperably linked 5′ or 3′ to additional enhancer elements that are inturn operably linked to a promoter which is operably linked 5′ to aleader element, operably linked to a coding sequence for 1-glucuronidase(GUS, GOI-Ec.uidA+St.LS1:1:1, SEQ ID NO:42) containing a processableintron derived from the potato light-inducible tissue-specific ST-LS1gene (Genbank Accession: X04753), operably linked to a 3′ terminationregion, and a left border region from A. tumefaciens (B-AGRtu.leftborder). The resulting plasmids are used to transform soybean plants orother genus plants by the methods described in the Examples.Alternatively, protoplast cells derived from soybean or other genusplants are transformed using methods known in the art to performtransient assays.

GUS expression driven by a regulatory element comprising one or moreenhancers is evaluated in stable or transient plant assays to determinethe effects of the enhancer element on expression of a transcribable DNAmolecule. Modifications to one or more enhancer elements or duplicationof one or more enhancer elements may be performed based upon empiricalexperimentation and the resulting gene expression regulation that isobserved using each regulatory element composition. Altering therelative positions of one or more enhancers in the resulting regulatoryor chimeric regulatory elements may affect the transcriptional activityor specificity of the regulatory or chimeric regulatory element and isdetermined empirically to identify the best enhancers for the desiredtransgene expression profile within the soybean plant or other genusplant.

Example 13 Analysis of the Effect Upon GUS Expression Imparted by theSynthetic 3′ UTR, T-Zm.GST7.Nno:2, in Stably Transformed Soybean Plants

Soybean plants were transformed with a vector, specifically plantexpression vectors containing regulatory element groups drivingexpression of the 1-glucuronidase (GUS) transgene. The resulting plantswere analyzed for GUS protein expression to assess the effect ofselected regulatory elements on expression.

Soybean plants were transformed with two binary vectors comprisingEXP-At.GSP571 (SEQ ID NO:4) driving GUS expression. The GUS transgenecassettes also comprised either the endogenous 3′ UTR T-Mt.Sali3-2-1:2:1(SEQ ID NO:34) or the synthetic 3′ UTR, T-Zm.GST7.nno:2 (SEQ ID NO:44).GUS protein expression was quantitatively measured in the organs ofstably transformed soybean plants transformed with the two constructs.Expression of GUS was compared between the constructs. Table 11 belowshows the mean GUS expression modulated by the synthetic 3′ UTR,T-Zm.GST7.nno:2, relative to the endogenous 3′ UTR, T-Mt.Sali3-2-1:2:1,wherein “nd” mean not determined and “bdl” means below detection limit.

TABLE 11 Mean quantitative GUS expression in stably transformed soybeanplants. Developmental T-Mt.Sali3-2-1:2:1 T-Zm.GST7.nno:2 Fold StageOrgan (SEQ ID NO: 34) (SEQ ID NO: 44) Attenuation V5 Root 40 bdlLeaf-Sink 650  88 7.4 Leaf-Source 1379 278 5.0 R1 Root 110  72 1.5Leaf-Petiole 951 199 4.8 Leaf-Source 1995 642 3.1 Flowers 703 139 5.1 R3Seed-Immature 75 bdl Pod 852 386 2.2 R5 Seed-Cotyledon 650 174 3.7 R8Seed-Embryo 1153 nd Seed-Cotyledon 2449 nd

As can be seen in Table 11, the synthetic 3′ UTR, T-Zm.GST7.nno:2attenuated expression relative to the 3′ UTR, T-Mt.Sali3-2-1:2:1 in alltissues assayed. The degree of attenuation varied for each tissue from1.5 fold in R1 Roots to 7.4 fold in V5 Sink Leaf. The use of a 3′ UTR toattenuate expression in stably transformed plants has great utility. Forexample, a 3′ UTR can be used in combination with other regulatoryelements such as promoters, leaders, and introns to fine tune expressionof a transgene, particularly those wherein high expression may lead tooff-phenotypic effects that are deleterious to the transformed plant.Analysis of the resulting GUS transcript confirmed proper termination ofthe transcript imparted by the synthetic 3′ UTR, T-Zm.GST7.nno:2. Thesynthetic 3′ UTR, T-Zm.GST7.nno:2, is able to modulate expression andproperly terminate transcription in stably transformed soybean plants.

Example 14 Analysis of the Synthetic 3′ UTRs, T-Zm.GST7.Nno:2 andT-Zm.GST59.Nno:1, on GUS Expression in Corn Protoplast Cells

Corn leaf protoplasts were transformed with vectors, specificallyexpression vectors containing test regulatory elements drivingexpression of the β-glucuronidase (GUS) transgene. The resultingtransformed corn leaf protoplasts were analyzed for GUS proteinexpression to assess the effect of the selected regulatory elements onexpression.

Corn protoplasts, derived from leaf tissue, were transformed withexpression vectors comprising synthetic expression elements and comparedto expression elements known in the art. Two expression vectors wereconstructed to assess the activity of the synthetic 3′ UTRs,T-Zm.GST7.nno:2 (SEQ ID NO:44) and T-Zm.GST59.nno:1 (SEQ ID NO:29) andtwo construct expression vectors were also constructed. Each of the fourconstructs comprised a transgene cassette comprising the constitutivepromoter and leader, EXP-CaMV.35S (SEQ ID NO:46), operably linked 5′ tothe intron I-Zm.DnaK:1 (SEQ ID NO:47), operably linked 5′ to a GUScoding sequence, GOI-Ec.uidA+St.LS1:1:1 (SEQ ID NO:42). The expressionvectors used to assess the synthetic 3′ UTRs comprised eitherT-Zm.GST7.nno:2 or T-Zm.GST59.nno:1 operably linked 3′ to the GUS codingsequence. One control vector comprised the 3′ UTR T-Os.LTP:1 (SEQ IDNO:48) operably linked 3′ to the GUS coding sequence. The other controlvector lacked a 3′ UTR.

A plasmid used in co-transformation of the protoplasts and normalizationof the data was also constructed using methods known in the art. Itcomprised a transgene cassette comprised of, EXP-CaMV.35S (SEQ ID NO:46)operably linked 5′ to a coding sequence encoding the NanoLuc® luciferasefluorescent protein (Promega, Madison, Wis. 53711), Nluc (SEQ ID NO:49),which was operably linked 5′ to a 3′ UTR, T-Os.LTP:1 (SEQ ID NO:48).

Corn leaf protoplasts were transformed using a PEG-based transformationmethod, similar to those known in the art. Protoplast cells weretransformed in a ninety six well format. Twelve micrograms of the testvector DNA or control vector DNA, and six micrograms of the NanoLuc®vector DNA were used to transform 3.2×10⁵ protoplasts per well. Aftertransformation, the protoplasts were incubated at 25° C. in the dark forsixteen to twenty hours. Following incubation, the protoplasts werelysed and the lysate used for measuring luciferase and GUS expression.To lyse the cells, the cells in the plate were pelleted throughcentrifugation, washed, resuspended in a smaller volume, and transferredto strip well tubes. The tubes were centrifuged again and supernatantwas aspirated leaving the protoplast cell pellet behind. The cell pelletwas resuspended in QB buffer (100 mM KPO₄, pH 7.8; 1 mM EDTA; 1% TritonX-100; 10% Glycerol; 1 mM DTT). The cells were lysed by vigorouslypipetting the cells several times, vortexing the tubes, and letting thetubes incubate on ice for five minutes. The lysate was then centrifugedto pellet the cell debris. The resulting lysate was then transferred toa clean plate.

Luciferase activity was assayed using the Nano-Glo® Luciferase AssaySubstrate (Promega, Madison, Wis. 53711) in QB buffer. In short, a smallvolume of lysate, QB buffer, and the Nano-Glo® Luciferase AssaySubstrate/QB solution were mixed together in white, ninety six wellplates. Fluorescence was then measured using a PHERAstar® plate reader(BMG LABTECH Inc., Cary, N.C. 27513).

GUS activity was assayed using the fluorogenic substrate4-methyleumbelliferyl-β-D-glucuronide (MUG) in a total reaction volumeof 50 microliters. The reaction product, 4-methlyumbelliferone (4-MU),is maximally fluorescent at high pH, where the hydroxyl group isionized. Addition of a basic solution of sodium carbonate simultaneouslystops the assay and adjusts the pH for quantifying the fluorescentproduct. An aliquot of lysate was mixed with an aliquot of MUG dissolvedin QB buffer and incubated at 37° C. A small aliquot of the lysate/MUGreaction mixture was removed and added to a stop buffer at threedifferent timepoints; (1) immediately after mixing the lysate/MUGreaction as “Time zero minutes”; (2) twenty minutes; and (3) sixtyminutes. Fluorescence was measured with excitation at 355 nm, emissionat 460 nm using a using a PHERAstar® plate reader (BMG LABTECH Inc.,Cary, N.C. 27513).

At least two plates were used in transformation with four to eighttransformations per plate for each expression vector. For each plate,each construct is transformed in four to eight wells. An aliquot istaken out of each transformation for the MUG assay and “nM MUGhydrolyzed” is derived from the in-plate-standard curve. An aliquot isalso taken out of each transformation for the NanoLuc® reading (NanoLuc®RLU). The mean nM MUG hydrolyzed/NanoLuc® RLU for each expression vectoris normalized with respect to the EXP-CaMV.35S/I-Zm.DnaK:1/T-Os.LTP:1expression vector which is set to 100%. Table 12 shows the average ofthe mean for all the plates used in transformation for each expressionvector comprising the synthetic 3′ UTRs T-Zm.GST7.nno:2 andT-Zm.GST59.nno:1, and the controls.

TABLE 12 Average of the mean nM MUG hydrolyzed/NanoLuc ® RLU for eachexpression vector. 3′ UTR Average of Mean Stderr T-Os.LTP:1 100.00 8.09No 3′ UTR 51.95 4.71 T-Zm.GST59.nno:1 505.45 37.75 T-Zm.GST7.nno:2345.31 40.73

As can be seen in Table 12, the expression vector without a 3′ UTRprovided less expression than the T-Os.LTP:1 control. Expression wasenhanced by the synthetic 3′ UTRs T-Zm.GST7.nno:2 and T-Zm.GST59.nno:1compared to the T-Os.LTP:1 control. Analysis of the transcriptsdemonstrated proper termination imparted by the synthetic 3′ UTRsT-Zm.GST7.nno:2 and T-Zm.GST59.nno:1. The synthetic 3′ UTRsT-Zm.GST7.nno:2 and T-Zm.GST59.nno:1 are able to modulate expression andproperly terminate transcription in transformed corn leaf protoplastcells.

Example 15 Analysis of Regulatory Elements Driving GUS in Cotton LeafProtoplasts

Cotton leaf protoplasts were transformed with vectors, specificallyexpression vectors containing regulatory element groups drivingexpression of the β-glucuronidase (GUS) transgene. The resultingtransformed cotton leaf protoplasts were analyzed for GUS proteinexpression to assess the effect of the selected regulatory elementgroups on expression.

Cotton protoplasts, derived from leaf tissue were transformed withexpression vectors comprising synthetic expression elements and comparedto expression elements known in the art. Separate experiments wereconducted to assess the activity of the EXP's, EXP-At.GSP571 (SEQ IDNO:4), EXP-At.GSP571.nno+At.GSI21.nno:10 (SEQ ID NO:8),EXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO:10),EXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO:16), andEXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO:22). The expressionelements were cloned into expression vectors and operably linked to aGUS coding sequence, GOI-Ec.uidA+St.LS1:1:1 (SEQ ID NO:42) thatcomprised a processable intron. The control expression vectors compriseddifferent configurations of known expression elements.

Two plasmids, for use in co-transformation and normalization of data,were also constructed using methods known in the art. Each plasmidcontained a specific luciferase coding sequence that was driven by aconstitutive EXP sequence. The plant vector pFLUC comprised a transgenecassette with a constitutive promoter operably linked 5′ to an intron,(EXP-CaMV.35S-enh+Zm.DnaK:1:1, SEQ ID NO: 53), operably linked 5′ to afirefly (Photinus pyralis) luciferase coding sequence (LUCIFERASE:1:3,SEQ ID NO: 54), operably linked 5′ to a 3′ UTR from the Agrobacteriumtumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 55).The plant vector pRLUC comprised a transgene cassette with aconstitutive EXP sequence (EXP-CaMV.35S-enh-Lhcb1, SEQ ID NO: 56),operably linked 5′ to a sea pansy (Renilla reniformis) luciferase codingsequence (CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO: 57), operably linked5′ to a 3′ UTR from the Agrobacterium tumefaciens nopaline synthase gene(T-AGRtu.nos-1:1:13, SEQ ID NO: 55).

Cotton leaf protoplasts were transformed using a PEG-basedtransformation method, known in the art. Protoplast cells weretransformed with the plasmids, pFLUC and pRLUC, and an equimolarquantity of the EXP expression vectors. Measurements of both GUS andluciferase were conducted by placing aliquots of a lysed preparation ofcells transformed as above into two different small-well trays. One traywas used for GUS measurements, and a second tray was used to perform adual luciferase assay using the dual luciferase reporter assay system(Promega Corp., Madison, Wis.; see for example, Promega Notes Magazine,No: 57, 1996, p.02). Sample measurements were based upon multipletransformations similar to that presented in Example 14. Mean GUS/FLUCvalues were calculated in a similar manner as in Example 14, but werenot normalized relative to the control EXP vectors.

The EXPs, EXP-At.GSP571 (SEQ ID NO:4), EXP-At.GSP571.nno+At.GSI21.nno:10(SEQ ID NO:8), and EXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO:10) werecloned into plant expression vectors operably linked 5′ to a GUS codingsequence (SEQ ID NO:42), operably linked 5′ to the 3′ UTR,T-Mt.Sali3-2-1:2:1 (SEQ ID NO:34). Two control plant expression vectorswere constructed with the EXP, EXP-At.Bglu21+At.Cyco:2 (SEQ ID NO:50),known to express poorly in cotton leaf protoplasts and the EXP,EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:51), known to express well incotton leaf protoplasts. The control EXPs were operably linked to thesame GUS and 3′ UTR sequence. In addition, a plant expression vectorcomprising a GUS transgene cassette comprising the EXP, EXP-At.GSP571(SEQ ID NO:4), operably linked to GUS comprised the synthetic 3′ UTR,T-Zm.GST7.nno:2 (SEQ ID NO:44) to assess the activity of the synthetic3′ UTR. The mean GUS/FLUC values for multiple transformations arepresented in Table 13.

TABLE 13 Mean GUS/FLUC values from transformed cotton leaf protoplastsEXP 3′ UTR Mean EXP SEQ ID NO: 3′ UTR SEQ ID NO: GUS/FLUCEXP-At.Bglu21 + 50 T-Mt.Sali3-2-1:2:1 34 0.09 At.Cyco:2EXP-CaMV.35S-enh + 51 T-Mt.Sali3-2-1:2:1 34 1.70 Ph.DnaK:1:3EXP-At.GSP571 4 T-Mt.Sali3-2-1:2:1 34 0.56 EXP-At.GSP571.nno + 8T-Mt.Sali3-2-1:2:1 34 1.02 At.GSI21.nno:10 EXP-At.GSP571.nno + 10T-Mt.Sali3-2-1:2:1 34 0.95 At.GSI102.nno:1 EXP-At.GSP571 4T-Zm.GST7.nno:2 44 0.46

As can be seen in Table 13, the EXPs, EXP-At.GSP571 (SEQ ID NO:4),EXP-At.GSP571.nno+At.GSI21.nno:2 (SEQ ID NO:8), andEXP-At.GSP571.nno+At.GSI102.nno:1 (SEQ ID NO: 10) demonstratedexpression in cotton leaf protoplast cells. The synthetic 3′ UTR,T-Zm.GST7.nno:10 (SEQ ID NO:44) functioned in a similar manner as theendogenous 3′ UTR, T-Mt.Sali3-2-1:2:1.

The EXP, EXP-At.GSP564.nno+At.GSI17.nno:2 (SEQ ID NO:16) was cloned intoa plant expression vectors operably linked 5′ to a GUS coding sequence(SEQ ID NO:42), operably linked 5′ to the endogenous 3′ UTR,T-Mt.Oxr-1:2:1 (SEQ ID NO:35). Two control plant expression vectors wereconstructed with the EXP, EXP-Gm.Sphas1:1:1 (SEQ ID NO:52), known toexpress poorly in cotton leaf protoplasts and the EXP,EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:51), known to express well incotton leaf protoplasts. The control EXPs were operably linked to thesame GUS and 3′ UTR sequence. The mean GUS/FLUC values for multipletransformations are presented in Table 14.

TABLE 14 Mean GUS/FLUC values from transformed cotton leaf protoplastsEXP 3′ UTR Mean EXP SEQ ID NO: 3′ UTR SEQ ID NO: GUS/FLUCEXP-Gm.Sphas1:1:1 52 T-Mt.Oxr-1:2:1 35 0.01 EXP-CaMV.35S-enh + 51T-Mt.Oxr-1:2:1 35 2.30 Ph.DnaK:1:3 EXP-At.GSP564.nno + 16 T-Mt.Oxr-1:2:135 0.34 At.GSI17.nno:2

As can be seen in Table 14, the synthetic EXP,EXP-At.GSP564.nno+At.GSI17.nno:1 (SEQ ID NO: 16) demonstrated expressionin cotton leaf cell protoplasts.

The EXP, EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO:22) was clonedinto a plant expression vectors operably linked 5′ to a GUS codingsequence (SEQ ID NO:42), operably linked 5′ to the endogenous 3′ UTR,T-Mt.RD22-1:2:1 (SEQ ID NO:37). Two control plant expression vectorswere constructed with the EXP, EXP-Gm.Sphas1:1:1 (SEQ ID NO:52), knownto express poorly in cotton leaf protoplasts and the EXP,EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:51), known to express well incotton leaf protoplasts. The control EXPs were operably linked to thesame GUS and 3′ UTR sequence. The mean GUS/FLUC values for multipletransformations are presented in Table 15.

TABLE 15 Mean GUS/FLUC values from transformed cotton leaf protoplastsEXP 3′ UTR Mean EXP SEQ ID NO: 3′ UTR SEQ ID NO: GUS/FLUCEXP-Gm.Sphas1:1:1 52 T-Mt.RD22-1:2:1 37 0.01 EXP-CaMV.35S-enh + 51T-Mt.RD22-1:2:1 37 2.88 Ph.DnaK:1:3 EXP-At.GSP579.nno + 22T-Mt.RD22-1:2:1 37 1.19 At.GSI102.nno:3

As can be seen in Table 15, the synthetic EXP,EXP-At.GSP579.nno+At.GSI102.nno:3 (SEQ ID NO:22), demonstratedexpression in cotton leaf cell protoplasts.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the claims. All publications and published patentdocuments cited herein are hereby incorporated by reference to the sameextent as if each individual publication or patent application isspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A recombinant DNA molecule comprising a DNAsequence selected from the group consisting of: a) a sequence with atleast 85 percent sequence identity to any of SEQ ID NOs:1-29 and 43-45;b) a sequence comprising any of SEQ ID NOs:1-29 and 43-45; and c) afragment of any of SEQ ID NOs:1-29 and 43-45, wherein the fragment hasgene-regulatory activity.
 2. The recombinant DNA molecule of claim 1,wherein the DNA sequence is operably linked to a heterologoustranscribable DNA molecule.
 3. The recombinant DNA molecule of claim 1,wherein the DNA sequence has at least 90 percent sequence identity tothe DNA sequence of any of SEQ ID NOs:1-29 and 43-45.
 4. The recombinantDNA molecule of claim 1, wherein the DNA sequence has at least 95percent sequence identity to the DNA sequence of any of SEQ ID NOs:1-29and 43-45.
 5. The recombinant DNA molecule of claim 1, wherein the DNAsequence comprises gene-regulatory activity.
 6. The recombinant DNAmolecule of claim 2, wherein the heterologous transcribable DNA moleculecomprises a gene of agronomic interest.
 7. The recombinant DNA moleculeof claim 6, wherein the gene of agronomic interest confers herbicidetolerance in plants.
 8. The recombinant DNA molecule of claim 6, whereinthe gene of agronomic interest confers pest resistance in plants.
 9. Atransgenic plant cell comprising the recombinant DNA molecule ofclaim
 1. 10. The transgenic plant cell of claim 9, wherein the DNAsequence is operably linked to a heterologous transcribable DNAmolecule.
 11. The transgenic plant cell of claim 9, wherein saidtransgenic plant cell is a monocotyledonous plant cell.
 12. Thetransgenic plant cell of claim 9, wherein said transgenic plant cell isa dicotyledonous plant cell.
 13. A transgenic plant, or part thereof,comprising the recombinant DNA molecule of claim
 1. 14. A progeny plantof the transgenic plant of claim 13, or a part thereof, wherein theprogeny plant or part thereof comprises the recombinant DNA molecule.15. A transgenic seed, wherein the seed comprises the recombinant DNAmolecule of claim
 1. 16. A method of producing a commodity productcomprising obtaining a transgenic plant or part thereof according toclaim 13 and producing the commodity product therefrom.
 17. The methodof claim 16, wherein the commodity product is protein concentrate,protein isolate, grain, starch, seeds, meal, flour, biomass, or seedoil.
 18. A method of expressing a transcribable DNA molecule comprisingobtaining a transgenic plant according to claim 13 and cultivatingplant, wherein the transcribable DNA is expressed.